IL-17A Promotes the Exacerbation of IL-33− Induced Airway Hyperresponsiveness by Enhancing Neutrophilic Inflammation via CXCR2 Signaling in Mice This information is current as of October 1, 2021. Nobuaki Mizutani, Takeshi Nabe and Shin Yoshino J Immunol 2014; 192:1372-1384; Prepublished online 20 January 2014; doi: 10.4049/jimmunol.1301538

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-17A Promotes the Exacerbation of IL-33–Induced Airway Hyperresponsiveness by Enhancing Neutrophilic Inflammation via CXCR2 Signaling in Mice

Nobuaki Mizutani,* Takeshi Nabe,† and Shin Yoshino*

Neutrophilic airway inflammation is a hallmark of patients with severe asthma. Although we have reported that both IL-33 and IL-17A contributed to IgE-mediated neutrophilic inflammation in mice, the relationship remains unclear. In this article, we examined how IL-17A modifies IL-33–induced neutrophilic inflammation and airway hyperresponsiveness (AHR). IL-33 was intratracheally administered to BALB/c mice on days 0–2; furthermore, on day 7, the effect of the combination of IL-33 and IL- 17A was evaluated. Compared with IL-33 or IL-17A alone, the combination exacerbated neutrophilic inflammation and AHR, associated with more increased levels of lung glutamic acid-leucine-arginine+ CXC , including CXCL1, CXCL2, and

CXCL5, and infiltration by alveolar expressing CXCR2. Treatment with anti-CXCR2 mAb or depletion of alveolar Downloaded from macrophages repressed neutrophilic inflammation and AHR; in addition, depletion of neutrophils suppressed AHR. These find- ings prompted us to examine the role of CXCR2 in IgE-sensitized mice; a single treatment with anti-CXCR2 mAb in the seventh Ag challenge inhibited late-phase airway obstruction, AHR, and neutrophilic inflammation. In addition to inhibition, multiple treatments during the fourth to seventh challenge attenuated early-phase airway obstruction, eosinophilic inflammation, and goblet cell hyperplasia associated with the reduction of Th2 production, including IL-4, IL-5, and IL-13. Collectively, IL-

33 cooperated with IL-17A to exacerbate AHR by enhancing neutrophilic inflammation via CXCR2 signaling; furthermore, http://www.jimmunol.org/ CXCR2 signaling derived Th2 responses. We thus suggest the underlying mechanisms of IL-33 and IL-17A in allergic asthma and CXCR2 as potential therapeutic targets for the disease. The Journal of Immunology, 2014, 192: 1372–1384.

llergic asthma is a chronic inflammatory disorder of the inflammatory pathways that have the potential to initiate and prop- airway associated with an increased level of allergen- agate allergic inflammation. However, the Th2 paradigm alone A specific IgE (1, 2). Although the unifying pathophysio- does not seem to explain the full spectrum of asthma, because logical features of asthma are allergen-induced early- and late- severe disease is not associated with an exclusive bias toward the phase asthmatic responses and airway hyperresponsiveness (AHR), production of Th2 (14, 15). by guest on October 1, 2021 which leads to airflow obstruction (2–4), the inflammatory status People with severe asthma have not only severe AHR and robust is complex and heterogeneous. For example, the airway granulo- neutrophils, but also produce IL-17A (14, 15), and experimental cytic infiltrate of most asthmatics in the lungs is dominated by mouse models of asthma suggest that IL-17A causes neutrophil eosinophils (2, 3, 5); however, severe asthma is characterized by recruitment and AHR (16, 17); therefore, it has been postulated predominantly neutrophilic lung inflammation (6, 7). Currently, that IL-17A production may drive severe forms of this disease. allergic models of asthma suggest that infiltration by eosinophils Meanwhile, glutamic acid-leucine-arginine (ELR)+ CXC chemo- arises through the production of CD4+ Th2-type cytokines, such as kines contribute to neutrophil migration, and seven human ELR+ IL-4, IL-5, and IL-13 in mice (8–10). Furthermore, IL-33 polar- CXC chemokines have been identified: growth-related oncogene- izes naive CD4+ T cells into a population of T cells that produce a (CXCL1), -b (CXCL2), and -g (CXCL3); epithelium-derived IL-5 (11) and induces the secretion of IL-5 and IL-13 from Th2 neutrophil-activating peptide 78 (CXCL5); chemo- cells (12, 13), suggesting that IL-33 promotes a number of key tactic protein 2 (CXCL6); neutrophil-activating peptide 2 (CXCL7); and IL-8 (CXCL8) (18). Among these chemokines, CXCL8 is the most potent neutrophil chemoattractant in humans, and its *Department of Pharmacology, Kobe Pharmaceutical University, Higashinada, Kobe analogs in mice include CXCL1 (keratinocyte-derived chemo- 658-8558, Japan; and †Department of Pharmacology, Kyoto Pharmaceutical Univer- sity, Misasagi, Yamashina, Kyoto 607-8414, Japan kine), CXCL2 (MIP-2), and CXCL5 (LPS-induced CXC chemo- Received for publication June 12, 2013. Accepted for publication December 18, kine); these chemokines, which act through the receptor CXCR2, 2013. appear to confer selectivity for promoting neutrophil migration This work was supported by Grant-in-Aid for Young Scientists (B) 23790164 (to N.M.) (18–22). In addition, administration of IL-17A to the lungs pro- and Grant-in-Aid for Scientific Research (C) 23590093 (to T.N.) from the Japan motes neutrophilic lung inflammation, as well as the expression of Society for the Promotion of Science. ELR+ CXC chemokines such as CXCL1 and CXCL2 in mice (23, Address correspondence and reprint requests to Dr. Nobuaki Mizutani, Depart- 24). More interestingly, there is a positive correlation between ment of Pharmacology, Kobe Pharmaceutical University, 4-19-1 Motoyamakita, + Higashinada, Kobe 658-8558, Japan. E-mail address: [email protected] increased expression of ELR CXC chemokines such as CXCL8 The online version of this article contains supplemental material. in humans and the presence of neutrophils in the lungs of severe Abbreviations used in this article: AHR, airway hyperresponsiveness; BALF, bron- asthmatics (25, 26), and a clinical study has reported the possi- choalveolar lavage fluid; ELR, glutamic acid-leucine-arginine; GR, glucocorticoid bility of CXCR2 as a potential therapeutic target in the disease receptor; ILC2, type 2 innate lymphocyte cell; MCh, methacholine; PAS, periodic (27); however, the functional significance and the pathways that acid–Schiff; sRaw, specific airway resistance. regulate the expression of their chemokines in the disease are not Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 fully defined. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301538 The Journal of Immunology 1373

We have previously reported that BALB/c mice passively sen- Passive sensitization with OVA-specific IgE mAb and treatment sitized with an i.p. injection of OVA-specific IgE mAb showed with mAbs early- and late-phase increases in airway resistance and AHR by OVA-specific IgE mAb (OE-1) was derived from a B cell hybridoma repeated intratracheal OVA challenges (28). In our previous (29, producing murine IgE, as described previously (28). The hybridoma was 30) and present studies, we demonstrated that the late-phase in- grown in CELLine CL1000 with BD-Cell-MAb medium (BD Biosciences, crease in airway resistance and AHR were closely associated with San Diego, CA) supplemented with 20% heat-inactivated FBS, 1% neutrophilic airway inflammation through IL-33 and IL-17A L-glutamine, and 1% penicillin-streptomycin. OE-1 levels in culture supernatants of hybridoma were assayed by ELISA (28). OE-1 levels were production. However, the precise relationship between IL-33 and calculated by comparison with mouse IgE standards (Southern Biotech, IL-17A remains unclear. Therefore, we investigated how IL-17A Birmingham, AL). modifies IL-33–induced neutrophilic inflammation and AHR, and Passive sensitization with OE-1 was performed with the protocol de- found that repeated intratracheal administration of IL-33 induced scribed previously (30). As shown in Fig. 6A, BALB/c mice were passively sensitized with repeated i.p. injections of a hybridoma supernatant con- neutrophilic inflammation and AHR, which were enhanced by the taining OE-1 (100 mg/mouse) on days 0, 1, and 2. Nonsensitized mice presence of IL-17A related to more increased levels of lung were injected with a culture supernatant of the parental myeloma cell line. CXCL1, CXCL2, and CXCL5 and alveolar macrophages ex- Both the sensitized and nonsensitized mice were challenged on days 1, 2, pressing CXCR2 in normal mice. Based on the data, we examined 3, 8, 9, 10, and 15 under isoflurane anesthesia with 1% OVA (grade V; the roles of CXCR2 signaling in neutrophilic inflammation and Sigma-Aldrich) in a volume of 20 ml by intratracheal administration. In addition, we have reported that purified OE-1 induced allergic airway in- AHR enhanced by the collaborative action of IL-33 and IL-17A in flammation by repeated Ag challenges (29). normal mice. In addition, in IgE-sensitized mice, the contribution A dose (20 mg/mouse) of anti-CXCR2 mAb or anti–IL-6 mAb was of CXCR2 signaling to airway inflammatory responses was in- administered intratracheally 30 min before the seventh Ag challenge in vestigated. OE-1–sensitized mice (Fig. 6A, single treatment). Furthermore, on days Downloaded from 8, 9, 10, and 15, a dose (20 mg/mouse) of anti-CXCR2 mAb or anti–IL-6 mAb was administered intratracheally 30 min before each of the Ag Materials and Methods challenges to mice sensitized with OE-1 (Fig. 6A, multiple treatments). Animals Control mice were given the same amount of rat IgG2a, rat IgG1, or rat IgG on days 8, 9, 10, and 15. Seven-week-old male BALB/c mice obtained from Japan SLC (Hamamatsu, In accordance with a previously reported method (29, 30), a dose (5 mg/

Japan) were used in this study to remove confounding effects of menstrual mouse) of anti–IL-33 mAb (R&D Systems) or rat IgG2a was administered http://www.jimmunol.org/ cycle–dependent changes on endogenous female sex hormones. These mice intratracheally 30 min before each of the challenges on days 8, 9, 10, and were maintained in a temperature-controlled environment with free access to 15 in mice sensitized with OE-1. Furthermore, a dose (150 mg/mouse) of standard rodent chow and water. All of the experimental procedures were anti–IL-17 mAb (R&D Systems) or rat IgG2a was administered i.p. 30 min approved by the Experimental Animal Research Committee at Kobe Phar- before the seventh challenge. maceutical University. Analysis of cells recovered by BAL Coadministration of IL-33 and IL-17A and treatment with mAbs, dexamethasone, or liposomal clodronate To evaluate airway cellular inflammation, we examined the accumulation of inflammatory cells in BALF as detailed previously (32). Animals were As shown Fig. 1A, we assessed the effect of coadministration of IL-33 and killed with diethyl ether. The trachea was cannulated and the left bronchi

IL-17A (Biolegend, San Diego, CA) on the accumulation of inflammatory were tied. The right air lumen was washed twice with 0.5 ml HBSS by guest on October 1, 2021 cells in bronchoalveolar lavage fluid (BALF) and AHR. IL-33 in solution containing 2% FBS. The cell pellet was suspended in a defined volume (200 ng/mouse) was administered intratracheally to normal mice on days (200 ml/sample) of HBSS containing 2% FBS. The total leukocyte count in 0, 1, and 2 under anesthesia with isoflurane (Wako Pure Chemicals, Osaka, the lavage fluid was determined by staining with Turk’s solution. For Japan) in a volume of 20 ml as reported previously (29). Furthermore, on differential cell counts, BAL cells were stained with Diff-Quik solution day 7, IL-33 (200 ng/mouse), IL-17A (40 ng/mouse), or IL-33+IL-17A (Sysmex International Reagents, Kobe, Japan). (200 + 40 ng/mouse) was administrated intratracheally, and the accumu- lation of inflammatory cells in BALF and AHR 24 h after the last instil- Measurement of AHR to methacholine lation was evaluated on day 8. To examine the effect of initial IL-33 treatments, we administered IL-33 (200 ng/mouse), IL-17A (40 ng/mouse), To estimate AHR to methacholine (MCh), we measured specific airway or IL-33+IL-17A (200 + 40 ng/mouse) intratracheally after pretreatment resistance (sRaw; cmH2O 3 mL/[mL/s]) before and after the intratracheal with PBS instead of IL-33. instillation of an MCh using a two-chambered, double-flow plethysmo- To evaluate the effects of anti-CXCR2 mAb, anti-CXCL1 mAb, anti- graph system (Pulmos-I; M.I.P.S., Osaka, Japan) according to the method CXCL2 mAb, or anti-CXCL5 mAb (R&D Systems, Minneapolis, MN) by Pennock et al. (33). In brief, increasingly higher doses of MCh (6.25, on the coadministration of IL-33– and IL-17–induced neutrophilic in- 12.5, and 25 mg/ml) in solution were administered consecutively via the flammation and AHR, we administered anti-CXCR2 mAb, anti-CXCL1 intratracheal route to mice under isoflurane-induced anesthesia at 30-min mAb, or anti-CXCL2 mAb (20 mg/mouse) intratracheally in a volume of intervals. sRaw was measured 2 min after the respective instillations of the 20 ml 30 min before coadministration; anti-CXCL5 mAb (60 mg/mouse) three doses of MCh solution. wasadministeredintratracheally3haftercoadministration,because CXCL5 production was not observed for at least 3 h after coadministration Measurement of airway resistance (Fig. 1). Control mice were given the same amount of rat IgG or rat IgG2b. In addition, a dose (20 mg/mouse) of anti–IL-6 mAb (Biolegend) or rat To evaluate the degree of early- and late-phase increases in airway resis- IgG1 was administered intratracheally 30 min before coadministration. tance, we measured sRaw before and 10 min to 5 h after the seventh Furthermore, in accordance with a previously reported method (29, 31), an challenge in IgE-sensitized mice. i.p. administered dose (150 mg/mouse) of anti–Gr-1 mAb (Biolegend) or rat IgG2b was given 18 h before coadministration to deplete neutrophils; Measurement of cytokines in lung tissue supernatants the dose of anti–Gr-1 mAb has been reported to fail to reduce the accu- mulation of other cells such as eosinophils and in BALF in The frozen right lobe (after BAL) was homogenized in 1 ml T-PER a murine model of asthma (31). (Thermo Scientific, Rockford, IL) containing a Complete Mini Protease Dexamethasone (3 mg/10 ml/kg; dexamethasone 21-phosphate disodium Inhibitor Cocktail tablet (Roche, Mannheim, Germany; 1 tablet/10 ml T- salt; Sigma-Aldrich, St. Louis, MO) was administered i.p. 2 h before co- PER stock reagent). Lung homogenates were centrifuged at 9000 3 g administration. In addition, to evaluate the role of alveolar macrophages, we for 10 min at 4˚C. CXCL1, CXCL2, CXCL5, IL-4, IL-5, IL-13 (R&D used a clodrosome depletion kit (Encapsula Nano Sciences, Systems), IL-17A, IL-33, and IL-6 (Biolegend) in supernatants of lung ho- Nashville, TN). Liposomal clodronate suspension (500 mg/mouse) was mogenates were measured using quantitative colormetric sandwich ELISA administered intratracheally in a volume of 100 ml 3 h after the coad- kits. The sensitivity of these assays was 2 pg/ml (CXCL1), 1.5 pg/ml ministration of IL-33 and IL-17A. Control mice were given the same (CXCL2), 0.347 pg/ml (CXCL5), 2 pg/ml (IL-4), 7 pg/ml (IL-5), 1.5 pg/ml amount of control liposome suspension. (IL-13), 2.7 pg/ml (IL-17A), 15.3 pg/ml (IL-33), and 2 pg/ml (IL-6). 1374 ROLE OF CXCR2 SIGNALING BY IL-33 AND IL-17A IN ASTHMA

Histological analysis a result, repeated intratracheal administration of IL-33 induced The left lungs were fixed in 10% neutral-buffered formalin, then dissected, neutrophilic inflammation and AHR, which were enhanced in embedded in paraffin, and cut 4 mm thick. Sections were stained with H&E combination with IL-17A compared with IL-33 or IL-17A alone– and periodic acid–Schiff (PAS). Scoring for each section was evaluated on treated mice (Fig. 1B, 1C); infiltration by alveolar macrophages a scale of 0 to 4 with increments of 0.5 by a blinded observer for in- and lymphocytes in IL-33+IL-17A–treated mice was increased in flammation (H&E) and goblet cell hyperplasia (PAS), as previously re- comparison with PBS-treated mice. In addition, sRaw values be- ported (32). fore the administration of MCh on day 8 were 2.258 6 0.077 in Flow cytometry analysis normal, 2.256 6 0.083 in PBS-treated, 2.217 6 0.093 in IL-33– 6 6 The whole lung was isolated, cut into 1-mm3 pieces in digestion buffer treated, 2.135 0.068 in IL-17A–treated, and 2.272 0.094 in (RPMI 1640 containing 150 U/ml collagenase [Wako Pure Chemicals], 30 IL-33+IL-17A–treated groups. Throughout the earlier experiment, mg/ml DNase I [Sigma-Aldrich], and 10 mM HEPES), and incubated at no significant differences in sRaw before administration were 37˚C for 1 h, as previously reported (30). The resulting single-cell sus- found among the groups. Meanwhile, there were no differences in pension was washed by centrifugation with PBS supplemented with 2% neutrophilic inflammation between IL-33 alone, IL-17A alone, or FBS, and cell numbers were determined using trypan blue staining after treatment with ACK lysis buffer to remove erythrocytes. IL-33+IL-17A with pretreatment with PBS instead of IL-33 Leukocytes recovered from collagenase/DNase I–digested lung tissue groups; in addition, neutrophilic inflammation by IL-33+IL-17A were incubated with anti-mouse FcgRII/III mAb followed by PerCP- without IL-33 pretreatment was very weak compared with that by Cy5.5–labeled anti-CXCR2 mAb, FITC-labeled anti-CD11c mAb, and PE- coadministration with IL-33 pretreatment (Table I). labeled anti-F4/80 mAb (Biolegend). After washing, the stained cells were + fixed with 4% paraformaldehyde and then analyzed using FACSCalibur ELR CXC chemokines, including CXCL1, CXCL2, and (BD Biosciences) and Cell Quest software (version 3.3; BD Biosciences). CXCL5 in mice, are a major subclass of chemokines involved in Downloaded from For detecting CXCR2+ cells in lung tissues, the cells were gated using side the recruitment of neutrophils (19–22). Therefore, we focused on scatter and CXCR2 expression; furthermore, in the cells, alveolar macro- the roles of these chemokines in AHR and neutrophilic inflam- phages were sorted based on their expression of F4/80 and CD11c, as mation induced by coadministration of IL-33 and IL-17A. Mean- shown in a previous report (34). In addition, to measure the absolute number of CXCR2+F4/80+CD11c+ cells in the lungs, the percentage of while, it has been reported that CXCL1 and CXCL2 CXCR2+F4/80+CD11c+ cells was multiplied by the number of total cells in production in the lungs peaked 3 h after the Ag challenge in the lungs. a murine model of asthma (22); in contrast, the level of CXCL5 in BALF peaked 24 h after the Escherichia coli challenge in mice http://www.jimmunol.org/ Measurement of OVA-specific IgE Ab in serum (35). Therefore, we examined whether coadministration of IL-33 OVA-specific IgE Ab in serum was measured by ELISA, as described and IL-17A exacerbated the production of these chemokines at 3 previously (28). OVA-specific IgE Ab in serum was detected using plates and 24 h in comparison with IL-33 or IL-17A alone–treated mice. coated with anti-mouse IgE Ab and adding biotin-labeled OVA. Streptavidin-HRP was added, the plate was developed with 3,39,5,59-tet- Significantly higher levels of CXCL1, CXCL2, and CXCL5 were ramethylbenzidine solution, and measurements were made at 450 nm using observed 24 h after the coadministration of IL-33 and IL-17A a microplate reader after stopping the reaction with sulfuric acid. Values compared with IL-33 or IL-17A alone–treated mice, although for serum OVA-specific IgE (1:5) were expressed as absorbance units. levels of these chemokines 3 h after administration showed no difference among the groups (Fig. 1D). Statistical analyses by guest on October 1, 2021 Data are shown as the mean 6 SEM. Statistical analyses between the two Treatment with anti-CXCR2 mAb inhibits neutrophilic groups were performed using Student t test (two-tailed). To compare more inflammation and AHR induced by coadministration of IL-33 than two groups, we used Dunnett’s test after conducting one-way ANOVA. and IL-17A A p value ,0.05 was considered significant. The results shown in Fig. 1 indicate that IL-17A enhances IL-33– Results induced neutrophilic inflammation and AHR associated with more increased production of ELR+ CXC chemokines. Therefore, we IL-17A enhances IL-33–induced neutrophilic inflammation and investigated the role of CXCR2 signaling in these IL-33 and IL- AHR associated with more increased levels of CXCL1, CXCL2, 17A coadministration-induced responses. The development of and CXCL5 neutrophilic inflammation and AHR induced by coadministration We have found that in IgE-sensitized mice, not only anti–IL-33 were strongly inhibited by anti-CXCR2 mAb; in addition, anti- mAb but also anti–IL-17A mAb decreased neutrophilic airway CXCL1 mAb, anti-CXCL2 mAb, and anti-CXCL5 mAb partially inflammation and AHR; anti–IL-33 mAb failed to inhibit IL-17A reduced them (Fig. 2A, 2B, 2D, 2E). Furthermore, anti-CXCR2 production in the lungs (29, 30) (Supplemental Fig. 1). In addition, mAb suppressed CXCL1, CXCL2, and CXCL5 production in the repeated intratracheal administration of IL-33 alone on days 0, 1, lungs; in addition, anti-CXCL1 mAb decreased CXCL2 and 2, and 7 did not induce IL-17A production in the lungs on day 8 CXCL5 production, although anti-CXCL2 mAb or anti-CXCL5 (PBS versus IL-33: 91.7 6 4.1 versus 97.8 6 4.9 pg/ml; n = 5). mAbfailedtoinhibitCXCL1andCXCL5orCXCL1andCXCL2, Meanwhile, the production of IL-33 in the lungs of IgE-sensitized respectively (Fig. 2C, 2F). In addition, anti-CXCL1 mAb, anti- mice was significant 24 h after the first and third challenges in CXCL2 mAb, and anti-CXCL5 mAb significantly decreased comparison with that in nonsensitized mice; a higher level was CXCL1, CXCL2, and CXCL5 levels in the lungs, respectively recognized 24 h after the fourth challenge. In addition, IL-17A (Fig. 2C, 2F). production was significant 24 h after the fourth challenge in com- parison with that in nonsensitized mice, although no production was Treatment with anti–IL-6 mAb inhibits neutrophilic seen 24 h after the first and third challenges (Supplemental Fig. 2). inflammation and AHR induced by coadministration of IL-33 Based on these data, we hypothesized that IL-33–induced neu- and IL-17A trophilic inflammation and AHR could be enhanced by the pres- It has been reported that IL-6 regulates neutrophil trafficking during ence of IL-17A; furthermore, the condition of IL-33–induced inflammatory responses (36). Therefore, we examined whether IL- airway inflammation may need to be considered in the interaction 6 contributes to neutrophilic inflammation and AHR induced by with IL-33 and IL-17A. Therefore, before the coadministration of coadministration of IL-33 and IL-17A. Although coadministration IL-33 and IL-17, IL-33 alone was administered repeatedly; as of IL-33 and IL-17A induced IL-6 production in the lungs, the The Journal of Immunology 1375 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021 FIGURE 1. IL-17A enhances IL-33–induced neutrophilic inflammation and AHR associated with more increased levels of CXCL1, CXCL2, and CXCL5. (A) Experimental protocol for intratracheal administration of IL-33 and IL-17A in BALB/c mice. IL-33 was administered intratracheally to normal mice on days 0, 1, and 2; furthermore, on day 7, PBS, IL-33, IL-17A, or IL-33+IL-17A was administrated intratracheally. (B) Changes in inflammatory cell numbers in BALF 24 h after the coadministration of IL-33 and IL-17A. (C) Changes in airway responsiveness to MCh 24 h after the coadministration of IL- 33 and IL-17A. (D) Changes in levels of CXCL1 (a), CXCL2 (b), and CXCL5 (c) in the lung tissue homogenates 3 and 24 h after the coadministration of IL-33 and IL-17A. Each value is the mean 6 SEM of 5–12 animals. *p , 0.05, **p , 0.01. Eos, Eosinophils; Lym, lymphocytes; Mac, macrophages; Neu, neutrophils; total, all cells. level was similar to IL-33 or IL-17A alone–treated mice (Fig. 3A). Depletion of alveolar macrophages inhibits neutrophilic However, IL-6 production was suppressed by anti-CXCR2 mAb inflammation and AHR induced by coadministration of IL-33 and anti-CXCL1 mAb, but not anti-CXCL2 mAb and anti-CXCL5 and IL-17A mAb (Fig. 3B). Furthermore, treatment with anti–IL-6 mAb As shown earlier, CXCL1, CXCL2, CXCL5, and IL-6 production, suppressed AHR and neutrophilic inflammation (Fig. 3C, 3D) which contribute to neutrophil recruitment, were produced via although CXCL1, CXCL2, and CXCL5 production in the lungs CXCR2 signaling (Figs. 2, 3). It has been reported that high levels was not decreased (Fig. 3E). of CXCL1 and CXCL2 induced by OVA challenge were observed in alveolar macrophages of mice sensitized with OVA (37). Based Table I. Comparison with neutrophilic airway inflammation induced by on this information, we hypothesized that chemokine and/or cy- intratracheal coadministration of IL-33 and IL-17A in mice pretreated tokine production may be induced via alveolar macrophages ex- with PBS or IL-33 pressing CXCR2 increased by coadministration of IL-33 and IL-17A. First, we assessed whether CXCR2 expression was in- No. of Cells (3105 Cells/BALF) creased on alveolar macrophages in the lungs of IL-33+IL-17A– treated mice. The absolute number of alveolar macrophages Total Cells Neutrophils (CD11c+F4/80+) expressing CXCR2 was calculated by multiply- PBS alone 1.65 6 0.06 0.06 6 0.02 ing by total cells in the lungs and percentages of the alveolar mac- 6 6 IL-33 (with PBS) 2.88 0.12 0.29 0.07 rophages expressing CXCR2; in addition, the number of total cells IL-17A (with PBS) 2.97 6 0.31 0.13 6 0.07 6 3 6 IL-33+IL-17A (with PBS) 2.98 6 0.25 0.33 6 0.09 in the lungs was 11.75 0.44 10 cells/lung in PBS-treated, IL-33+IL-17A (with IL-33) 16.72 6 0.57* 8.54 6 0.20* 13.36 6 0.74 3 106 cells/lung in IL-33–treated, 14.93 6 0.75 3 6 6 3 6 Each value is the mean 6 SEM of four animals. 10 cells/lung in IL-17A–treated, and 24.64 2.36 10 cells/ *p , 0.01 compared with the IL-33+IL-17A (with PBS) group. lung in IL-33+IL-17A–treated groups. Percentages and absolute 1376 ROLE OF CXCR2 SIGNALING BY IL-33 AND IL-17A IN ASTHMA Downloaded from http://www.jimmunol.org/

FIGURE 2. Treatment with anti-CXCR2 mAb inhibits neutrophilic inflammation and AHR induced by coadministration of IL-33 and IL-17A. (A, B, D,

and E) Effects of anti-CXCR2 mAb, anti-CXCL1 mAb, anti-CXCL2 mAb, and anti-CXCL5 mAb on neutrophilic inflammation (A and D) and AHR (B and E) by guest on October 1, 2021 24 h after the coadministration of IL-33 and IL-17A. (C and F) Effects of anti-CXCR2 mAb, anti-CXCL1 mAb, anti-CXCL2 mAb, and anti-CXCL5 mAb on the production of CXCL1 (a), CXCL2 (b), and CXCL5 (c) in the lung tissue homogenates 24 h after the coadministration of IL-33 and IL-17A. Each value is the mean 6 SEM of 5–12 animals. *p , 0.05, **p , 0.01; #p , 0.05, ##p , 0.01 compared with IL-33+IL-17A rat IgG or rat IgG2b group. Eos, Eosinophils; Lym, lymphocytes; Mac, macrophages; Neu, neutrophils; total, all cells. cell numbers of alveolar macrophages (CD11c+ F4/80+) express- treatment with anti–Gr-1 mAb suppressed AHR in IL-33+IL- ing CXCR2 in the lungs of IL-33 alone–, IL-17A alone–, and IL- 17A–treated mice (Fig. 5A, 5B); furthermore, this treatment sup- 33+IL-17A–treated groups were increased in comparison with the pressed IL-6 production in the lungs, but not CXCL1, CXCL2, PBS-treated group (Fig. 4A). Furthermore, the absolute cell and CXCL5 (Fig. 5C). Meanwhile, steroids are considered one of numbers of alveolar macrophages (CD11c+ F4/80+) expressing the most effective treatments for asthma. Therefore, we assessed CXCR2 in the IL-33+IL-17A–treated group were increased the effect of dexamethasone on neutrophilic inflammation and compared with the IL-33 or IL-17A alone–treated group, although AHR. As shown in Fig. 5D–F, neutrophilic inflammation, AHR, the percentages did not change among the groups (Fig. 4A). and CXCL1, CXCL2, CXCL5, and IL-6 production were not Therefore, we investigated the effects of the depletion of alveolar suppressed by this treatment. macrophages by liposomal clodronate on neutrophilic inflamma- Treatment with anti-CXCR2 mAb or anti–IL-6 mAb inhibits tion, AHR, and the production of ELR+ CXC chemokine and IL-6 IgE-mediated airway inflammatory responses induced by coadministration of IL-33 and IL-17A. The treatment inhibited neutrophilic inflammation and AHR; furthermore, CXCL1 In IgE-sensitized mice, treatment with anti–IL-33 mAb or anti–IL- and CXCL2 production in the lungs was suppressed, but not CXCL5 17A mAb inhibited CXCL1, CXCL2, and CXCL5 production in and IL-6 (Fig. 4B–D). In addition, the number of alveolar mac- the lungs 24 h after the seventh Ag challenge (Table II). Therefore, rophages was significantly reduced in the IL-33+IL-17A–clodro- we examined whether CXCR2 signaling is also important for the nate–treated group (2.61 6 0.41 3 105 cells/BALF, p , 0.05) in development of IgE-mediated airway inflammatory responses. comparison with the IL-33+IL-17A vehicle–treated group (4.75 6 Treatment with a single dose or multiple doses of anti-CXCR2 0.36 3 105 cells/BALF). mAb significantly suppressed neutrophilic accumulation in BALF, the late-phase increase in airway resistance, and AHR; in addition, Depletion of neutrophils inhibits AHR induced by CXCL1, CXCL2, and CXCL5, but not IL-6, were decreased by coadministration of IL-33 and IL-17A this treatment (Fig. 6B–E). Furthermore, multiple treatments in- Subsequently, we examined the role of neutrophils in AHR and hibited the early-phase increase in airway resistance and eosino- ELR+ CXC chemokine and IL-6 production, because neutrophils philic inflammation, although the single treatment did not show highly expressed CXCR2 (38). The depletion of neutrophils by inhibitory effects on these responses (Fig. 6B, 6C). In addition, The Journal of Immunology 1377 Downloaded from http://www.jimmunol.org/

FIGURE 3. Treatment with anti–IL-6 mAb inhibits neutrophilic inflammation and AHR induced by the coadministration of IL-33 and IL-17A. (A) Change in level of IL-6 in the lung tissue homogenates 24 h after the coadministration of IL-33 and IL-17A. (B) Effects of anti-CXCR2 mAb, anti-CXCL1 mAb, anti-CXCL2 mAb, and anti-CXCL5 mAb on the production of IL-6 in the lung tissue homogenates 24 h after the coadministration of IL-33 and IL- 17A. (C and D) Effects of anti–IL-6 mAb on neutrophilic inflammation (C) and AHR (D) 24 h after the coadministration of IL-33 and IL-17A. (E) Effects of anti–IL-6 mAb on the production of CXCL1 (a), CXCL2 (b), and CXCL5 (c) in the lung tissue homogenates 24 h after the coadministration of IL-33 and IL-17A. Each value is the mean 6 SEM of 4–10 animals. *p , 0.05, **p , 0.01; #p , 0.05, ##p , 0.01 compared with IL-33+IL-17A rat IgG1 group. Eos, by guest on October 1, 2021 eosinophils; Lym, lymphocytes; Mac, macrophages; Neu, neutrophils; total, all cells. throughout the earlier experiment, sRaw values before the seventh matics, particularly those who have severe asthma (6, 40, 41) and challenge were 2.484 6 0.036 in the nonsensitized challenged, are resistant to corticosteroids (42), have raised neutrophil counts 2.505 6 0.050 in the IgE-sensitized challenged rat IgG2a-treated, in their airways, suggesting neutrophils to be a more valid target 2.508 6 0.007 in the IgE-sensitized challenged anti-CXCR2 mAb than eosinophils in certain stages of the pathogenesis. Meanwhile, single-treated, and 2.515 6 0.024 in the IgE-sensitized challenged although we have found that not only IL-33 but also IL-17A-se- anti-CXCR2 mAb multiple-treated groups, indicating that no sig- creting Th17 cells contributed to IgE-mediated neutrophilic in- nificant differences in sRaw before the challenge were found among flammation (29, 30), the relationship between IL-33 and IL-17A the groups. Meanwhile, treatment with a single dose or multiple has not yet been defined. In this study, we focused on how IL-17A doses of anti–IL-6 mAb showed similar inhibitory effects of anti- modifies IL-33–induced neutrophilic inflammation and AHR; as CXCR2 mAb on the biphasic increase in airway resistance, AHR, a result, repeated intratracheal administration of IL-33 induced and neutrophilic and eosinophilic inflammation (Fig. 7A–C). Al- neutrophilic inflammation and AHR, which were enhanced in though single treatment with anti–IL-6 mAb did not suppress combination with IL-17A associated with more increased levels of CXCL1, CXCL2, and CXCL5 production, multiple treatments in- lung CXCL1, CXCL2, and CXCL5 and alveolar macrophages hibited only CXCL1 production (Fig. 7D). expressing CXCR2. Treatment with anti-CXCR2 mAb or deple- Furthermore, airway inflammation (H&E) and goblet cell hy- tion of alveolar macrophages suppressed neutrophilic inflamma- perplasia (PAS) in the lungs were inhibited by multiple treatments tion and AHR; in addition, depletion of neutrophils inhibited AHR, with anti-CXCR2 mAb or anti–IL-6 mAb (Fig. 8A, 8B). In ad- suggesting the importance of CXCR2 signaling to regulate the co- dition, multiple treatments with anti-CXCR2 mAb or anti–IL-6 operative action of IL-33– and IL-17A–enhanced AHR via neu- mAb suppressed OVA-specific IgE Ab (Fig. 9A), IL-5, and IL-13 trophilic inflammation. Based on these data, we attempted to (Fig. 9B); anti-CXCR2 mAb, but not anti–IL-6 mAb, inhibited examine the role of CXCR2 in IgE-sensitized mice; a single IL-4 production (Fig. 9B). treatment with anti-CXCR2 mAb at the seventh Ag challenge inhibited late-phase airway obstruction, AHR, and neutrophilic Discussion inflammation. Thus, we propose the mechanism that IL-17A ex- Allergic asthma has long been considered an eosinophilic bron- acerbates IL-33–induced AHR by enhancing neutrophilic inflam- chitis; however, its symptoms were not alleviated by a marked mation via CXCR2 signaling in allergic asthma. reduction in eosinophil numbers in blood and the airway by Treatment with anti–IL-33 mAb or anti–IL-17A mAb inhibited treatment with anti–IL-5 in clinical cases (39, 40). Some asth- neutrophilic inflammation and AHR in IgE-sensitized mice (29, 1378 ROLE OF CXCR2 SIGNALING BY IL-33 AND IL-17A IN ASTHMA Downloaded from http://www.jimmunol.org/

FIGURE 4. Depletion of alveolar macrophages inhibits neutrophilic inflammation and AHR induced by the coadministration of IL-33 and IL-17A. (A) by guest on October 1, 2021 Representative dot plots depicting the percentages of alveolar macrophages expressing CXCR2 (CXCR2+CD11c+F4/80+) in the lungs 24 h after the co- administration of IL-33 and IL-17A (a). Changes in the number of CXCR2+CD11c+F4/80+ cells in the lungs of PBS, IL-33, IL-17, and IL-33+IL-17 groups (b). (B and C) Effects of liposomal clodronate on neutrophilic inflammation (B) and AHR (C) 24 h after the coadministration of IL-33 and IL-17A. (D) Effects of liposomal clodronate on the production of CXCL1 (a), CXCL2 (b), CXCL5 (c), and IL-6 (d) in the lung tissue homogenates 24 h after the coadministration of IL-33 and IL-17A. Each value is the mean 6 SEM of 4–10 animals. *p , 0.05, **p , 0.01; #p , 0.05, ##p , 0.01 compared with IL- 33+IL-17A vehicle group. Eos, Eosinophils; Lym, lymphocytes; Mac, macrophages; Neu, neutrophils; total, all cells.

30) (Supplemental Fig. 1). Therefore, we hypothesized that the Thus, initial IL-33–responsive Th2 cells and ILC2s may elicit interaction with IL-33 and IL-17A may enhance neutrophilic in- changes that conditioned the mice to a quantitatively different flammation and AHR. Furthermore, IL-33 expression in the lungs response as a consequence of the coadministration of IL-33 and was observed during the first to fourth Ag challenges, although IL- IL-17A. Meanwhile, consistent with previous data (29), in which 17 was recognized only in the fourth challenge in IgE-sensitized the depletion of neutrophils inhibited AHR in IgE-sensitized mice, mice (Supplemental Fig. 2), suggesting that the condition of IL- the development of AHR induced by the coadministration of IL- 33–induced airway inflammation may need to be considered in the 33 and IL-17A was also inhibited by the depletion, suggesting that interaction with IL-33 and IL-17A. Therefore, before the coad- the increased numbers of neutrophils in the lungs are associated ministration of IL-33 and IL-17, IL-33 alone was administered with the development of AHR. The finding that activated neu- repeatedly; as a result, repeated intratracheal administration of trophils release a large array of inflammatory mediators, oxygen IL-33 induced neutrophilic inflammation and AHR, which were radicals, and protease has lent support to the notion of their in- strongly enhanced by the presence of IL-17A (Fig. 1). In addition, volvement in the intense inflammation found in severe asthma as expected, there was no enhancement of neutrophilic inflam- (5, 45). mation by coadministration of IL-33 and IL-17A without IL-33 It has been reported that ELR+ CXC chemokines such as pretreatments compared with IL-33 or IL-17A alone–treated mice CXCL1, CXCL2, and CXCL5 mainly contribute to neutrophilic (Table I). In the previous study (30), we found that repeated ad- inflammation in mice (19–22). Therefore, we examined the rela- ministration of IL-33 alone induced airway inflammation related tionship between IL-33 and IL-17A in the production of CXCL1, to Th2 cytokine production in the lungs, such as IL-4, IL-5, and CXCL2, and CXCL5. Repeated intratracheal administration of IL- IL-13. Several cell types, including CD4+ Th2 cells and type 2 33 induced CXCL1, CXCL2, and CXCL5, which were enhanced innate lymphocyte cells (ILC2s) producing Th2 cytokines, are by the presence of IL-17A (Fig. 1), suggesting the possibility that targets for IL-33; indeed, IL-33–responsive adaptive immune more increased ELR+ CXC chemokine production induced by IL- cells, such as allergen-specific Th2 cells, and innate cells, such as 33 and IL-17A may enhance the development of AHR via neu- ILC2s, cooperate to induce allergic airway inflammation (43, 44). trophilic inflammation. Therefore, we investigated the role of The Journal of Immunology 1379 Downloaded from http://www.jimmunol.org/

FIGURE 5. Depletion of neutrophils inhibits AHR and IL-6 production induced by the coadministration of IL-33 and IL-17A. (A and B) Effects of anti– Gr-1 mAb on neutrophilic inflammation (A) and AHR (B) 24 h after the coadministration of IL-33 and IL-17A. (C) Effects of anti–Gr-1 mAb on the production of CXCL1 (a), CXCL2 (b), CXCL5 (c), and IL-6 (d) in the lung tissue homogenates 24 h after the coadministration of IL-33 and IL-17A. (D and E) Effects of dexamethasone on neutrophilic inflammation (D) and AHR (E) 24 h after the coadministration of IL-33 and IL-17A. (F) Effects of dexamethasone on the production of CXCL1 (a), CXCL2 (b), CXCL5 (c), and IL-6 (d) in the lung tissue homogenates 24 h after the coadministration of IL-33 and IL-17A. Each value is the mean 6 SEM of four to five animals. *p , 0.05, **p , 0.01; #p , 0.05, ##p , 0.01 compared with IL-33+IL-17A rat IgG2b. Eos, Eosinophils; Lym, lymphocytes; Mac, macrophages; Neu, neutrophils; total, all cells. by guest on October 1, 2021 CXCR2 in AHR and neutrophilic inflammation. Treatment with mAbfailedtoinhibitCXCL1andCXCL5orCXCL1andCXCL2, anti-CXCR2 mAb suppressed neutrophilic inflammation and AHR respectively, both treatments suppressed AHR and neutrophilic in- in mice treated with IL-33 and IL-17A (Fig. 2). Thus, the coop- flammation (Fig. 2). Thus, these data suggest that CXCL1-CXCR2 erative action of IL-33 and IL-17A exacerbates AHR by enhan- signaling controls CXCL2 and CXCL5 production, which con- cing neutrophilic inflammation induced by CXCR2 signaling. tributes to neutrophilic inflammation and AHR. Meanwhile, it has Furthermore, we examined the detailed interaction with CXCL1, been reported that IL-6 induces neutrophilic inflammation (36). CXCL2, and CXC5 in neutrophilic inflammation and AHR en- Consistent with this finding, treatment with anti–IL-6 mAb re- hanced by IL-33 and IL-17A. Interestingly, treatment with anti- duced neutrophilic inflammation and AHR (Fig. 3); furthermore, CXCR2 mAb decreased CXCL1, CXCL2, and CXCL5 produc- IL-6 production was inhibited by treatment with anti-CXCR2 tion in the lungs; in addition, anti-CXCL1 mAb suppressed AHR mAb or anti-CXCL1 mAb. Thus, IL-6 regulated by CXCL1- and neutrophilic inflammation, as well as CXCL2 and CXCL5 CXCR2 signaling also contributed to neutrophilic inflammation production (Fig. 2). Although anti-CXCL2 mAb or anti-CXCL5 and AHR. However, anti–IL-6 mAb had no effect on ELR+

Table II. Effects of anti–IL-33 mAb or anti–IL-17A mAb on CXCL1, CXCL2, and CXCL5 production in the lung 24 h after the seventh Ag challenge in IgE-sensitized mice

CXCL1 CXCL2 CXCL5 Anti–IL-33 mAb NS-C (7th) 0.09 6 0.01 0.06 6 0.01 0.62 6 0.03 OE-1 (7th) + rat IgG2a 0.78 6 0.11* 0.52 6 0.05* 2.43 6 0.20* OE-1 (7th) + anti–IL-33 0.50 6 0.03** 0.27 6 0.08** 1.43 6 0.13** Anti–IL-17A mAb OE-1 (7th) + rat IgG2a 0.67 6 0.04 0.34 6 0.04 2.23 6 0.14 OE-1 (7th) + anti–IL-17A 0.43 6 0.05** 0.22 6 0.03** 1.39 6 0.11** Values are in ng/ml. Anti–IL-33 mAb was intratracheally administered on days 8, 9, 10, and 15 (OE-1 [seventh] + anti–IL- 33). Negative and positive controls were nonsensitized challenged (NS-C [seventh]) and OE-1–sensitized challenged, control rat IgG2a mAb-treated (OE-1 [seventh] + rat IgG2a) mice, respectively. Furthermore, anti–IL-17A mAb was i.p. administered on day 15 (OE-1 [seventh] + anti–IL-17A). Positive control was OE-1–sensitized challenged, control rat IgG2a mAb-treated (OE-1 [seventh] + rat IgG2a) mice. Each value is the mean 6 SEM of 5–11 animals. *p , 0.01 compared with the NS-C (seventh) group, **p , 0.05 compared with the OE-1 (7th) + rat IgG2a group. 1380 ROLE OF CXCR2 SIGNALING BY IL-33 AND IL-17A IN ASTHMA Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021 FIGURE 6. Treatment with anti-CXCR2 mAb inhibits IgE-mediated airway inflammatory responses. (A) Experimental protocol for sensitization with OE-1 and challenge with Ag, and treatment with anti-CXCR2 mAb. A single treatment with anti-CXCR2 mAb was intratracheally administered on day 15 to IgE-sensitized mice. In addition, multiple treatments with anti-CXCR2 mAb were intratracheally administered on days 8, 9, 10, and 15. Negative and positive controls were nonsensitized challenged and IgE-sensitized challenged, control rat IgG2a mAb-treated mice, respectively. (B–E) Effects of treatment with a single dose or multiple doses of anti-CXCR2 mAb on inflammatory cells in BALF (B), biphasic increase in airway resistance (C), AHR (D), and CXCL1 (a), CXCL2 (b), CXCL5 (c), or IL-6 production (d) in the lung tissue homogenates (E). Each value is the mean 6 SEM of 5–12 animals. *p , 0.05, **p , 0.01; ¥p , 0.05, ¥¥p , 0.01; or #p , 0.05, ##p , 0.01 compared with the NS-C (seventh) or OE-1 (seventh)+rat IgG2a group, respectively. Eos, Eosinophils; Lym, lymphocytes; Mac, macrophages; Neu, neutrophils; total, all cells.

CXC chemokines, including CXCL1, CXCL2, and CXCL5, pro- neutrophilic inflammation and AHR, as well as CXCL1 and duced by coadministration of IL-33 and IL-17A; coadministration CXCL2 production in the lungs (Fig. 4), indicating the possibility of IL-33 and IL-17A did not induce greater production of IL-6 in the that the infiltration of alveolar macrophages expressing CXCR2 lungs. It has been reported that IL-6 suppressed neutrophil apoptosis after coadministration plays a critical role in neutrophilic in- dependent on the increased number of neutrophils (46). Based on flammation and AHR through CXCL1 and CXCL2 production. these findings, it is suggested that ELR+ CXC chemokine produc- However, the inhibitory levels of CXCL1 and CXCL2 in the lungs tion enhanced by IL-33 and IL-17A strongly induces neutrophilic by the depletion of alveolar macrophages were weak, although inflammation, and IL-6 exhibits prolonged survival of neutrophils. neutrophilic inflammation was strongly inhibited. It has been re- Furthermore, in this study, the depletion of neutrophils inhibited IL- ported that activated alveolar macrophages produce TNF-a and/or 6 production (Fig. 5), indicating that neutrophils may inhibit their IL-1b to express adhesion molecules such as ICAM-1 and E- own apoptosis through IL-6 production. selectin, which contribute to neutrophil migration through the To define the cells important for coadministration of IL-33– and endothelium (47, 48), suggesting that, in this study, depletion of IL-17A–induced ELR+ CXC chemokine production via CXCR2, alveolar macrophages may repress the recruitment of neutrophils we assessed the potential of alveolar macrophages because they through decreased expressions of not only CXCL1 and CXCL2, are important sources of CXCL1 and CXCL2 (37). In this study, but also adhesion molecules. Meanwhile, the depletion of alveolar the number of alveolar macrophages expressing CXCR2 was in- macrophages and neutrophils did not inhibit CXCL5 production creased after the coadministration of IL-33 and IL-17A in com- (Figs. 4, 5). It has been reported that this chemokine is upregulated parison with IL-33 or IL-17A alone–treated mice (Fig. 4), in lung epithelial and endothelial cells during inflammation (49), suggesting that IL-17A enhances IL-33–induced alveolar macro- suggesting that epithelial and endothelial cells might contribute to phages expressing CXCR2. Furthermore, the depletion of alveolar the production of CXCL5 enhanced by the cooperative action of macrophages by treatment with liposomal clodronate suppressed IL-33 and IL-17A. The Journal of Immunology 1381

FIGURE 7. Treatment with anti–IL-6 mAb inhibits IgE-mediated airway in- flammatory responses. (A–D) Effects of treatment with a single dose or multiple doses of anti–IL-6 mAb on inflamma- tory cells in BALF (A), biphasic increase in airway resistance (B), AHR (C), and CXCL1 (a), CXCL2 (b), and CXCL5 (c) in the lung tissue homogenates (D). Each value is the mean 6 SEM of five to six animals. *p , 0.05, **p , 0.01; #p , 0.05, ##p , 0.01, compared with the OE-1 (seventh)+rat IgG1 group. Eos, Eosinop- Downloaded from hils; Lym, lymphocytes; Mac, macro- phages; Neu, neutrophils; total, all cells. http://www.jimmunol.org/

It has been reported that Ag-induced AHR, neutrophilic in- ELR+ CXC chemokine production, indicating that AHR and flammation, and ELR+ CXC chemokine production, such as neutrophilic inflammation exacerbated by IL-33 and IL-17A were CXCL1 and CXCL2 in experimental asthmatic models associated also steroid resistant. Exposure of epithelial cells to IL-17A with IL-17A using Th17 cell–reconstituted SCID and RORgt-tg in vitro significantly increased glucocorticoid receptor b (GRb), by guest on October 1, 2021 mice, were steroid resistant (50, 51). Consistent with these data, which is thought to act as a dominant negative inhibitor of GRa, we found that dexamethasone failed to inhibit coadministration of the classical GR (52); indeed, bronchial biopsies from individuals IL-33– and IL-17A–induced AHR, neutrophilic inflammation, and with asthma showed increased expression of IL-17A and decreased

FIGURE 8. Multiple treatments with anti-CXCR2 mAb or anti–IL-6 mAb inhibit IgE-mediated airway inflammation and goblet cell hyperplasia. (A and B) Effects of multiple treatments with anti-CXCR2 mAb or anti–IL-6 mAb on IgE-mediated airway inflammation (H&E) (A) and goblet cell hyperplasia (PAS) (B) in OE-1 (7th)+rat IgG (a), OE-1 (7th)+anti-CXCR2 mAb (b), or OE-1 (7th)+anti–IL-6 mAb groups (c). Scale bars, 100 mm. Histological appearance 24 h after the seventh challenge was scored for inflammation and goblet cell hyperplasia (d). Each value is the mean 6 SEM of 4–10 animals. *p , 0.05, **p , 0.01. 1382 ROLE OF CXCR2 SIGNALING BY IL-33 AND IL-17A IN ASTHMA

FIGURE 9. Multiple treatments with anti-CXCR2 mAb or anti–IL-6 mAb in- hibit IgE-mediated Ag-specific IgE Ab and Th2 cytokine production. (A and B) Effects of multiple treatments with anti- CXCR2 mAb or anti–IL-6 mAb on IgE- mediated endogenous Ag-specific IgE Ab production in the serum (A) and Th2 cytokine production including IL-4 (a), IL-5 (b), and IL-13 (c) in the lung tissue homogenates (B). Each value is the mean 6 SEM of 4–12 animals. *p , 0.05, **p , 0.01. Downloaded from expression of GRa (53), suggesting that IL-17A may directly inflammation and goblet cell hyperplasia were exacerbated during induce steroid insensitivity. Meanwhile, IL-17A production in- these challenges (30). Interestingly, the infiltration of eosinophils duced by an Ag challenge was not inhibited by dexamethasone in and goblet cell hyperplasia was suppressed by multiple treatments Th17 cell–reconstituted SCID mice (50); in contrast, we found with anti-CXCR2 mAb in IgE-sensitized mice (Figs. 6, 8). It is that IL-33 production in the lungs was inhibited by dexametha- known that Th2 cytokines, including IL-5 and IL-13, particularly sone in OVA+alum-sensitized/OVA-challenged mice (T. Nabe, contribute to eosinophilic inflammation and goblet cell hyperplasia, http://www.jimmunol.org/ N. Mizutani, and S. Yoshino, unpublished observations), and pre- respectively (8–10); in this study, Th2 cytokine production, in- treatment with IL-33 was needed to induce changes in the inter- cluding IL-5 and IL-13, were inhibited by the treatment (Fig. 9), action with IL-33 and IL-17A, indicating that the reduction of IL-33 suggesting that CXCR2 signaling induced these IgE-mediated production by steroids may regulate responses such as AHR, neu- responses through Th2 immune responses. Meanwhile, it has trophilic inflammation, and ELR+ CXC chemokine production in been reported that IL-6 contributes to Th2 immune responses (56). experimental asthmatic models associated with IL-33 and IL-17A. In addition to the inhibitory effects of anti-CXCR2 mAb, multiple To clarify the importance of CXCR2 signaling in allergic airway treatments with anti–IL-6 mAb also inhibited eosinophilic in- responses, we first investigated the effects of anti–IL-33 mAb or flammation and goblet cell hyperplasia associated with the re- + anti–IL-17A mAb on the production of ELR CXC chemokines in duction of Th2 cytokine production such as IL-5 and IL-13 by guest on October 1, 2021 IgE-sensitized mice; these mAbs suppressed CXCL1, CXCL2, (Figs. 7–9). However, anti–IL-6 mAb showed a slight inhibi- and CXCL5 production in the lungs (Table II), suggesting that IL- tion of Th2 cytokine production, especially in IL-13; in contrast, 33 and IL-17A contributed to IgE-mediated ELR+ CXC chemo- IL-6 directly induced goblet cell hyperplasia (57), indicating that kine production. Next, we examined the effect of anti-CXCR2 in addition to IL-13, goblet cell hyperplasia in IgE-sensitized mAb on IgE-mediated airway inflammatory responses. In mice mice may be induced directly by IL-6. sensitized with IgE, a single treatment with anti-CXCR2 mAb at Multiple treatments with anti-CXCR2 mAb or anti–IL-6 mAb the seventh challenge inhibited late-phase airway obstruction, AHR, inhibited early-phase airway obstruction (Figs. 6, 7). Furthermore, neutrophilic inflammation, and ELR+ CXC chemokine production both multiple treatments inhibited the production of Ag-specific (Fig. 6), suggesting that CXCR2 signaling contributes to the de- IgE at the seventh challenge in IgE-sensitized mice (Fig. 9). Our velopment of IgE-mediated airway inflammatory responses. Mean- previous report showed that the level of Ag-specific IgE was sig- while, a single treatment with anti–IL-6 mAb also suppressed late- nificantly increased over the injection levels of IgE (OE-1) alone phase airway obstruction, AHR, and neutrophilic inflammation in (28). Based on these data, the suppression of the early-phase increase IgE-sensitized mice (Fig. 7). However, IL-6 production was not in airway resistance by these treatments likely contributed to the inhibited by anti-CXCR2 mAb in IgE-sensitized mice (Fig. 6), al- reduction of endogenous Ag-specific IgE Ab in IgE-sensitized mice. though production was inhibited by the mAb in IL-33+IL-17A– In conclusion, we demonstrated that: 1) IL-17A exacerbates treated mice. Thus, the mechanism of IL-6 production in IgE- IL-33–induced neutrophilic inflammation and AHR associated mediated airway inflammation was not congruent with that in with enhanced ELR+ CXC chemokines in the lungs and alveolar the cooperative action of IL-33 and IL-17A, suggesting that in our macrophages expressing CXCR2; 2) CXCR2 signaling regulates IgE-sensitized model, IL-6 production may occur by other the cooperative action of IL-33– and IL-17A–enhanced AHR via mechanisms independent of the CXCR2 signaling induced by IL- neutrophilic inflammation; and 3) in IgE-sensitized mice, CXCR2 33 and IL-17A, because there are more complex mechanisms in signaling plays essential roles in the development of airway in- IgE-mediated airway inflammation. flammatory responses. Published works have suggested that anti- It is widely known that eosinophilic inflammation and goblet cell IgE therapy is effective for some patients with poorly controlled hyperplasia are characteristic features of asthma associated with asthma (58–60). On the basis of our results, the importance of Th2 responses (8–10, 54, 55). It is of interest to examine whether CXCR2 signaling by the interaction with IL-33 and IL-17A in CXCR2 signaling also contributes to these responses. In this study, IgE-mediated allergic asthma associated with neutrophilic in- we examined the effect of multiple treatments with anti-CXCR2 flammation may provide insight into the underlying pathogenesis of mAb during the fourth to seventh challenges on eosinophilic chronic inflammatory disease, in which IgE has an important inflammation and goblet cell hyperplasia, because eosinophilic pathogenic role. The Journal of Immunology 1383

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