IL-13 receptor α1 differentially regulates aeroallergen induced lung responses

doi:10.4049/jimmunol.1004159

J Immunol. Author manuscript; available in PMC 2012 November 1.

Published in final edited form as:

J Immunol. 2011 November 1; 187(9): 4873–4880.

Published online 2011 September 28. doi: 10.4049/jimmunol.1004159

PMCID: PMC3197875

NIHMSID: NIHMS322622

IL-13 receptor α1 differentially regulates aeroallergen induced lung responses

Marc E Rothenberg,^*² Ting Wen,^*³ Dana Shik,^†³ Eric T Cole,^* Melissa M Mingler,^* and Ariel Munitz^†^2,¹

^*Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH, 45229

^†Department of Microbiology and Clinical Immunology, The Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel

² Address correspondence to Ariel Munitz, PhD, Department of Clinical Microbiology and Immunology, The Sackler School of Medicine, The Tel-Aviv University, 69978, Israel, Phone: 972-3-640-7636, Fax: 972-3-640-7635, Email: arielm/at/post.tau.ac.il and/or Marc E. Rothenberg, MD, PhD, Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave, MLC 7028, Cincinnati, OH, 45229., Phone: 513-803-0257, Fax: 513-636-3310, Email: Rothenberg/at/cchmc.org

¹This study was supported by grants to A.M and M.E.R from US-Israel Binational Science Foundation (2009222); to A.M. from the FP7 Marie-Curie Reintegration grant; to M.E.R. from NIH AI83450, AI045898, DK076893, the Buckeye Foundation, the Food Allergy Project/Food Allergy Initiative, and the Campaign Urging Research For Eosinophilic Disease (CURED).

³Contributed equally.

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Abstract

Interleukin (IL)-13 and IL-4 are hallmark cytokines of Th2-associated diseases including asthma. Recent studies revealed that IL-13Rα1 regulates asthma pathogenesis by mediating both IL-4 and IL-13-mediated responses. Nonetheless, the relative contribution of each cytokine in response to aeroallergen challenge and the degree of functional dichotomy between IL-4 and IL-13 in asthma remains unclear. Consistent with prior publications, we demonstrate that IL-13Rα1 regulates aeroallergen-induced airway resistance and mucus production but not IgE and Th2 cytokine production. We demonstrate that aeroallergen-induced eosinophil recruitment and chemokine production were largely dependent of IL-13Rα1 following Aspergillus (Asp) but not house dust mite (HDM) challenges. Notably, Asp-challenged mice displayed increased IL-13Rα1-dependent accumulation of dendritic cell subsets into lung draining lymph nodes in comparison with HDM. Comparison of IL-4 and IL-13 levels in the different experimental models revealed increased IL-4:IL-13 ratios following HDM challenge, likely explaining the IL-13Rα1-independent eosinophilia and chemokine production. Consistently, eosinophil adoptive-transfer experiments revealed near ablation of lung eosinophilia in response to Asp in Il13ra1^−/− mice, suggesting that Asp-induced lung eosinophil recruitment is regulated by IL-13-induced chemokine production, rather than altered IL-13 signaling in eosinophils. Furthermore, the near complete protection observed in Il13ra1^−/− mice in response to Asp-challenge was dependent on mucosal sensitization since Alum/Asp-sensitized mice that were re-challenged with Asp developed IL-13Rα1-independent eosinophilia although other asthma parameters remained IL-13Rα1-dependent. These results establish that IL-13Rα1 is required for aeroallergen-induced airway resistance and that allergen-induced chemokine production and consequent eosinophilia is dictated by the balance between IL-4 and IL-13 production in situ.

Keywords: Allergy, Lung, Cytokines, Chemokines, Cytokine receptors, Eosinophils

Introduction

Interleukin (IL)-13 is a hallmark T helper type 2 cytokine that mediates central characteristics of allergic asthma including IgE synthesis, mucus hypersecretion, airway hyperreactivity and fibrosis (1). The biological functions of IL-13 largely overlap with IL-4 (1, 2), being explained by common usage of the IL-4Rα chain in both IL-4- and IL-13-induced signaling. IL-4 mediates its effects either through the type I IL-4 receptor (R), composed of the IL-4Rα and common γ chains, or the type II IL-4R, composed of the IL-4Rα and IL-13Rα1 chains. Adding complexity to the functions of these two cytokines in Th2 settings is the differential expression of the unique receptor chains (i.e. the common γ and IL-13Rα1 chains) in distinct cells, which renders them either IL-4-responsive/IL-13-non-responsive (type I IL-4) or IL-4-responsive/IL-13-responsive (type II IL-4R) (1, 3). For example, airway epithelial cells do not express the common γ chain and thus respond to IL-4 via the type II IL-4R whereas myeloid cells express both the type I and type II IL-4R and can thus be activated by IL-4 and IL-13 (4, 5). Strategies targeting IL-13Rα1 for anti-asthma therapy are currently underway (6); however, there is incomplete data regarding the role of IL-13rα1 in response to naturally occurring aeroallergens, which often trigger asthma.

Recent studies have demonstrated a key role for IL-13Rα1 and the type II IL-4R in lung Th2 responses (3, 7). We have previously identified IL-13Rα1 as a fundamental receptor mediating IL-13- and IL-4-induced AHR, mucus production and fibrosis in response to the “classical” experimental asthma model using OVA/alum sensitization followed by lung OVA-challenge (3). Nevertheless, the role of IL-13α1 in experimental asthma models of natural occurring clinically relevant aeroallergen sensitization and mucosal challenge is unknown. This is especially noteworthy since eosinophil recruitment to the lung following adjuvant sensitization (alum) and consequent allergen exposure (OVA) is predominantly IL-13Rα1 independent even though eosinophil-selective chemokine expression (CCL11, CCL24) is entirely dependent on IL-13Rα1 (3).

Since IL-4 and IL-13 are co-upregulated in the lungs following allergen challenge (8, 9), it is likely that their differential expression and/or upregulation may determine the dependency of the asthmatic response on IL-13Rα1. Supporting this hypothesis, we have shown that IL-13Rα1 regulates both IL-4 and IL-13 signaling in the lung. While IL-13-dependent responses were entirely dependent on IL-13Rα1, IL-4-induced chemokine production and inflammatory cell recruitment were IL-13Rα1 independent (3).

In the study presented herein, we further establish the fundamental role of IL-13Rα1 in allergen-induced airway resistance, mucus production and TGF-β induction. We reveal that lung chemokine expression and consequent eosinophil accumulation are differentially dependent on IL-13Rα1 and determined by allergen type and route of sensitization, which dictates the balance between IL-4 and IL-13. Furthermore, we demonstrate that dendritic cell accumulation in lung draining lymph nodes is mediated by IL-13Rα1-dependent and -independent pathways differentially regulated by specific aeroallergens.

Materials and Methods

Mice

Generation of Il13ra1^−/− mice has been previously described (3, 7). Mice were backcrossed into their respective strains (BALB/c and C57BL/6) for at least 10 generations. For all experiments, BALB/c or C57BL/6 wild type mice were obtained from Charles River (Wilmington, MA) and housed under specific pathogen-free conditions. The institutional animal experimentation ethics committee approved all of the experiments.

Allergen sensitization and challenge

Asp and HDM antigen-associated asthma was induced by challenging mice intranasally three times a week for 3 weeks as previously described (5, 10, 11). In brief, mice were lightly anesthetized with isoflurane inhalation, and 10 μg of total protein (and not dry weight) in 50 μl saline of Asp or HDM extract (Bayer Pharmaceuticals, Spokane, Washington, USA) or 50 μl of normal saline solution alone was applied to the nasal cavity by using a micropipette with the mouse held in the supine position. After instillation, mice were held upright until alert. Mice were euthanized 24–48 hours after the last challenge. In some experiments, asthma models were induced by two intraperitoneal injections with 100 μg Asp extract and 1 mg aluminum hydroxide (alum) as adjuvant (14 days apart), followed by two intranasal challenges of 50 μg Asp extract or saline (3 days apart), starting a least 10 days after the second sensitization, as previously described (10, 11). The level of LPS in the Asp and HDM extracts was less than 2 pg/ml as detected by the Limulus assay. Mice were sacrificed 24–48 hrs after the last intranasal challenge.

Immunoglobulin and mediator assessment

Serum IgE and bronchoalveolar lavage fluid (BALF) cytokines were measured with kits purchased from the followingsources: IgE (BD bioscience, Lower detection limit: 15 pg/ml); and CCL11, CCL24, CCL2, CCL17, IL-4, IL-13, IL-5 and active TGF-β (R&D Systems, Lower detection limits: 15.62, 32.25, 15.62, 32.5, 3.91, 31.25, 6.25 and 31.25 pg/ml, respectively).

Real-time PCR

RNA samples from the whole esophagus were subjected to reverse transcription analysis using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) according to manufacturer’sinstructions. Real-time PCR analysis of Il13, Il4 and Hprt levels was performed using the LightCycler 480 system inconjunction with the ready-to-use LightCycler 480 SYBR Green I Master reaction kit (Roche Diagnostic Systems, Branchburg,NJ). Results were normalized to Hprt cDNA (12, 13).

Airway resistance and compliance measurements

Airway resistance was measured using the flexiVent system (Scireq Scientific Respiratory Equipment Inc) (3). Briefly, the mice were anesthetized, a tracheotomy was performed and a cannula inserted. A positive end-expiratory pressure (PEEP) of 0.2 kPa was established. Saline aerosol followed by β-methylcholine (Sigma-Aldrich, MO, USA; 25 and 50 mg/ml, BALB/c and C57BL/6, respectively) established control baseline. Aerosols were generated with an ultrasonic nebulizer (DeVilbiss UltraNeb 2000, Somerset, PA, USA) and delivered to the inspiratory line of the flexiVent. Each aerosol was delivered for 20 seconds during which time regular ventilation was maintained. Five measurements were made at 25-second intervals, and 3 peak responses were compared to the mean response of the saline aerosol.

Lung histopathology and immunohistochemistry

Histological studies were performed as follows: the right upper lobe of saline- or allergen-challenged lungs was fixed in 3.7%paraformaldehyde, embedded in paraffin, deparaffinized, and stained with hematoxylin and eosin or with periodic acid – Schiff (PAS) reagent (14). PAS-stained slides were quantified as previously described (3, 11). Lung and esophageal eosinophils were stained and quantified by immunohistochemistry as described previously (14, 15).

Flow cytometry

Forty-eight hours following the last aeroallergen-challenge the mice were sacrificed and lung draining lymph nodes were harvested. Lymph nodes were delicately crushed to generate single cell suspensions. Thereafter, single cell suspensions we stained with the following antibodies (all purchased from eBioscience): CD45-605NC, CD11c-Alexa Flour 488, CD11b-APC, B220-PE, Gr-1-PE-Cy7 and acquired by the Gallios flow cytometer (Beckman Coulter). Data analysis was performed using Kaluza (Beckman Coulter) or FloJow (TreeStar) on at least 50,000 events.

Adoptive transfer experiments

Eosinophils were grown from the bone marrow (BM) of wild type mice with modifications based on a prior report (16). Briefly, BM cells were harvested and loaded on a histopaque gradient (Sigma). Low-density BM cells were collected and cultured in the presence of SCF and Flt3L for four days. Thereafter, the medium was replaced with IL-5 for the rest of the culture (up to day 16) (16). On days 14–16 of the BM culture, 8 × 10⁶ eosinophils were injected into the tail vein of Asp-challenged mice (8 hrs after the 5–6^th allergen challenge). Bronchoalveolar lavage fluid was extracted 48 hours after the transfer.

Statistical analysis

Data were analyzed by ANOVA followed by Tukey post hoc test using GraphPad Prism 4 (San Diego, CA). Data are presented as mean ± SD, and values of p < 0.05 were considered statistically significant.

Results

Regulation of Asp-induced airway resistance, compliance and mucus production in Il13ra1^−/− mice

To define the role of IL-13Rα1 in the response to naturally occurring airborne allergens, we subjected Il13ra1^−/− mice to intranasal exposure to Asp, a potent inducer of allergic airway inflammation (17–19). Assessment of airway resistance in response to cholinergic stimuli revealed that Il13ra1^−/− mice were entirely protected from the allergen-induced increases in airway resistance observed in wild type mice (Figure 1A). Furthermore, Il13ra1^−/− mice had a concomitant protection from allergen-induced reductions in airway compliance (Figure 1B). To examine the role of IL-13Rα1 in allergen-induced mucus production, histological sections of Asp-challenged lungs were stained with PAS, and PAS⁺ cells were enumerated. Il13ra1^−/− mice were entirely protected from allergen-induced mucus production and goblet cell hyperplasia (Figure 1C–D). Assessment of active TGF-β levels in Asp-challenged Il13ra1^−/− mice demonstrated markedly reduced TGF-β levels in comparison with Asp-challenged wild type (Figure 1E).

Figure 1

Regulation of Asp-induced airway resistance, compliance and mucus production in Il13ra1^−/− mice

Regulation of Asp-induced lung chemokine production and leukocytosis in Il13ra1^−/− mice

IL-13 and IL-4 are potent inducers of various chemokines including CCL11, CCL24 and CCL17. To define the role of IL-13Rα1 in aeroallergen-induced chemokine production, BALF from Asp-challenged wild type and Il13ra1^−/− mice was examined for the aforementioned chemokines. Il13ra1^−/− mice displayed nearly complete protection from Asp-induced CCL11 and CCL24 expression (93% and 91% reduction, respectively; Figure 2A-B). Moreover, Asp-induced CCL17 was undetectable in BALF samples obtained from Il13ra1^−/− mice (Figure 2C). Consistent with the substantial decrease in chemokine expression, cellular recruitment of eosinophils into the BALF and lungs was dramatically attenuated in allergen-challenged Il13ra1^−/− mice (Figure 2D); however, no changes were observed in neutrophil and lymphocyte BALF levels. Lung tissue eosinophila was decreased by ~80% in Asp-challenged Il13ra1^−/− mice (Figure 2E). To determine whether IL-13Rα1 regulates eosinophilia in a tissue-specific fashion, we assessed Asp-induced eosinophilia in the esophagus (20). Notably, aeroallergen-challenged Il13ra1^−/− mice displayed near complete protection from eosinophil accumulation into the esophagus as well (Figure 2F). To definitely demonstrate that decreased eosinophil migration into the lungs of Asp-challenged Il3ra1^−/− mice was not due to an intrinsic defect of IL-13 signaling in eosinophils, an adoptive transfer approach was employed. Wild type eosinophils, generated from low-density bone marrow cells, were adoptively transferred intravenously into Asp-challenged wild type and Il13ra1^−/− mice. Indeed, donor wild type eosinophils that were adoptively transferred into Asp-challenged wild type mice were readily detectable in the BAL (Figure 2G). In sharp contrast, wild type eosinophils that were transferred into Asp-challenged Il13ra1^−/− mice were hardly detectable in the BAL and markedly reduced (100–1000 fold lower than in wild type mice) (Figure 2G).

Figure 2

Regulation of Asp-induced lung chemokine production and leukocytosis in Il13ra1^−/− mice

Assessment of Asp-induced IgE and Th2 cytokine induction in Il13ra1^−/− mice

The striking protection of Il13ra1^−/− mice from the local effects of Asp suggested that Il13ra1^−/− might not be able to mount a typical Th2 response, which is characterized by increased IgE production and expression of hallmark Th2 cytokines such as IL-4, IL-13 and IL-5 (21, 22). To examine this possibility, Asp-challenged wild type and Il13ra1^−/− mice were assessed for total serum IgE. No difference was observed in allergen-induced total serum IgE levels between wild type and Il13ra^−/− mice (Figure 3A). Consistent with this observation, Asp-challenged Il13ra1^−/− mice displayed a significant increase in IL-4, IL-13 and IL-5 levels (Figure 3B–D). Interestingly, Il13ra1^−/− mice displayed a minor, but statistically significant, increase in IL-13 levels (Figure 3C) in comparison with wild type mice but had similar IL-4 and IL-5 levels (Figures 3B and 3D).

Figure 3

Assessment of IgE production and Th2 cytokines in Asp-challenged Il13ra1^−/− mice

Regulation of HDM-induced airway resistance, compliance and mucus production in Il13ra1^−/− mice

Various studies have demonstrated different mechanisms for allergenicity to airborne allergens (23–25). Thus, we were interested to examine whether the roles of IL-13Rα1 in the regulation of allergen-induced lung responses were allergen specific or a shared phenomenon between allergens. To address this question, we employed an additional model of mucosal sensitization following repetitive HDM intranasal exposures. Similar to our findings with Asp-challenged Il13ra1^−/− mice (Figure 1), HDM-challenged Il13ra1^−/− mice were entirely protected from increased allergen-induced airway resistance and decreased compliance (Figure 4A–B). Assessment of PAS⁺ cells in Il13ra1^−/− mice following HDM challenge revealed that allergen-challenged Il13ra1^−/− mice displayed nearly complete protection from allergen-induced mucus production (Figure 4C–D). Furthermore, HDM-challenged mice were also protected from allergen-induced elevation in TGF-β (Figure 4E).

Figure 4

Regulation of Asp-induced lung chemokine production and leukocytosis in Il13ra1^−/− mice

Regulation of HDM-induced lung chemokine production and leukocytosis in Il13ra1^−/− mice

Interestingly, and in contrast to our findings in Il13ra1^−/− mice following Asp-challenge, HDM-challenged Il13ra1^−/− mice displayed elevated CCL11, CCL24 and CCL17 levels in the BALF, albeit decreased compared with wild-type mice (Figure 5A–C). These results were confirmed by real-time (q) PCR analysis (data not shown) demonstrating only partial regulation of CCL11, CCL24 and CCL17 production by IL-13Rα1 in response to HDM challenge. Consistent with these findings, HDM-challenged Il13ra1^−/− mice revealed substantial eosinophil infiltration into the lungs and BALF of HDM-challenged mice, which was predominantly IL-13Rα1 independent (Figure 5D–E). Similar to our findings with Asp, HDM-challenged Il13ra1^−/− mice displayed similar IgE and Th2 cytokines compared with HDM-challenged wild type mice (Figure 6A–D).

Figure 5

Regulation of HDM-induced lung chemokine production and leukocytosis in Il13ra1^−/− mice

Figure 6

Assessment of IgE production and Th2 cytokines in HDM-challenged Il13ra1^−/− mice

Differential IL-4 and IL-13 production in response to Asp and HDM

We have previously shown that IL-13Rα1 differentially regulates IL-4- and IL-13-induced responses in the lung (3). Thus, we hypothesized that the role of IL-13Rα1 in response to allergen challenge may be dictated by the net ratio between allergen-induced IL-4 and IL-13. Comparing the key roles of IL-13Rα1 in Asp-induced chemokine production and eosinophil recruitment (Figures 1–2) with its partial role in HDM-induced chemokine production and eosinophil recruitment suggested that HDM may preferentially utilize the type I IL-4R as the ratio of IL-4 to IL-13 should be higher following HDM-challenge than after Asp-challenge. To investigate this possibility, qPCR analysis of saline- and allergen-challenged (Asp and HDM) lungs obtained from wild type mice was performed. Indeed, both Asp and HDM were capable of significantly increasing IL-4 and IL-13 mRNA expression (Figure 7A–D). To determine relative IL-4 and IL-13 levels in the different models, allergen-induced IL-4 and IL-13 mRNA levels were normalized to IL-4 and IL-13 baseline levels in saline-treated mice (Figure 7E). Notably, IL-4:IL-13 mRNA ratios in HDM-induced responses were higher than those observed in response to Asp challenge (Figure 7F).

Figure 7

Differential IL-4 and IL-13 production in response to Asp and HDM

Differential regulation of dendritic cell accumulation in lung draining lymph nodes by IL-13Rα1

Dendritic cells (DCs) have key roles in the initiation of Th2 responses by regulating the polarization of Th2 cells and thus IL-4 and IL-13 cytokine production. Hence, we next hypothesized that IL-13Rα1 may differentially regulate recruitment of DCs into lung draining lymph nodes (LDLN) in response to allergen challenge. Assessment of B220⁺/CD11b⁻/Gr-1⁺ and B220⁻/CD11b⁺/Gr-1⁻ DC subsets following Asp and HDM challenge revealed significantly increased DC accumulation into the LDLN (Figure 8A–B). Interestingly, IL-13Rα1 predominantly regulated DC accumulation in LDLN in response to Asp-challenge and to a significantly lesser extent following HDM-challenge (Figure 8C–D).

Figure 8

Differential regulation of dendritic cell accumulation in lung draining lymph nodes by IL-13Rα1

Alum sensitization leads to IL-13Rα1-independent allergen-induced eosinophilic inflammation in the lung

Given the striking IL-13Rα1 dependency of eosinophilia following Asp challenge, we aimed to define whether this phenomenon was attributed to the mode of allergic sensitization or an inherent trait of the allergen itself. Therefore, we established a model of experimental airway inflammation using alum and Asp similar to the conventional alum and OVA model (10, 11) and assessed allergen-induced lung inflammation. As expected, Il13ra1^−/− mice were entirely protected from increased allergen-induced airway resistance and decreased allergen-induced compliance (Figure 9A–B), mucus production and TGF-β expression (Figure 9C–D). Furthermore, allergen-induced chemokine (e.g. CCL11, CCL24 and CCL17) production was entirely dependent on IL-13Rα1 (Figure 9E–G). However, lung eosinophilia was predominantly independent of IL-13α1 as Il13ra1^−/− mice displayed similar eosinophil levels to wild type mice (Figure 9H–I).

Figure 9

Effect of sensitization on allergen-induced IL-13Rα1-independent lung eosinophilic inflammation

Discussion

The pathological effects of IL-4 and IL-13 in Th2 immunity have been a focus of intense research in the last decade (1, 4, 10, 26). Indeed, both cytokines are capable of driving major features of allergic asthma, namely airway resistance, mucus production and fibrosis. Thorough examination of the IL-4/IL-13:IL-13Rα1 signaling axis in asthma requires further attention as agents that target these cytokines, receptors and subsequent signaling responses are being actively developed for the treatment of Th2-associated diseases especially asthma. To fully dissect the involvement of IL-13Rα1 in the lung, we examined diverse Th2 responses in Il13ra1^−/− mice following mucosal sensitization and challenge of natural occurring, clinically relevant aeroallergens, namely Asp and HDM. We report that a) IL-13Rα1 is the key receptor mediating airway hyperresponsiveness, mucus production and TGF-β induction in response to aeroallergens; b) Decreased eosinophilia in Asp-challenged Il13ra1^−/− mice is not due to a defect in IL-13 signaling in eosinophils but due to extrinsic activity likely mediated by IL-13Rα1-regulated chemokine production; c) The dependency of eosinophil recruitment into the lungs following allergen challenge is dictated by the relative ratios of allergen-induced IL-4 and IL-13; d) Dendritic cell accumulation in the lung draining lymph nodes in response to aeroallergens is differentially regulated by IL-13Rα1 following diverse allergen challenge which may account for the distinct differences in the IL-4:IL-13 ratios; e) IL-13Rα1 is required for allergen-induced esophageal eosinophilia; and f) finally, we demonstrate that unlike its role in schistosoma egg antigen-induced airway inflammation (27), in response to aeroallergens, IL-13Rα1 does not mediate an inhibitory Th2 cytokine network/balance.

One of the major findings presented herein is that following Asp challenge, eosinophil recruitment and chemokine production are largely IL-13Rα1-dependent. Indeed, our adoptive transfer experiments indicate that eosinophil recruitment to the lungs following Asp-challenge is likely regulated by IL-13-induced chemokine production likely by epithelial cells rather than an inherent defect in IL-13-induced responses of eosinophils. In fact, we cannot demonstrate direct signaling induced by IL-13 on murine eosinophils, even though IL-4 is very potent (manuscript in preparation). In contrast, following HDM challenges IL-13Rα1-independent pathways exist, which regulate eosinophil recruitment and chemokine production. The finding that alum/Asp-sensitized Il13ra1^−/− mice developed substantial lung eosinophilia independent of IL-13Rα1 indicates that the mode of allergen sensitization is a key determinant for IL-13Rα1 dependency. Moreover, we show that allergen-induced esophageal eosinophilia is IL-13Rα1-dependent. This finding is particularly important since IL-13 has been shown to be sufficient to induce eosinophilic esophagitis in mice (28) and likely man (29, 30). Yet, the receptor requirement has not been elucidated even though Il13ra2^−/− mice display increased esophagitis (31). Since anti-IL-13 reagents are now in clinical trials for asthma and eosinophilic esophagitis, these pre-clinical findings have broad implications. Mechanistically, we demonstrate increased IL-4:IL-13 ratios following HDM challenge; this may explain the IL-13Rα1-independent eosinophilia and chemokine production as IL-4 likely becomes the more dominant signaling pathway under these conditions. This suggests that in allergic settings where IL-13 production is relatively higher than IL-4, blockade of IL-13Rα1 will have better therapeutic value than in allergic settings displaying lower IL-4 to IL-13 ratios. In low IL-4:IL-13 ratios, observation of IL-4-driven chemokine production and tissue eosinophilia may be likely. It is notable that Asp and HDM utilize distinct mechanisms to induce allergic lung inflammation; HDM exerts its effects via functional mimicry of Toll-like receptor signaling (24, 32, 33); whereas, Asp utilizes protease dependent pathways (23, 34, 35). Exposure of airway epithelium to HDM results in upregulation of CCL20, which attracts immature dendritic cells. Interestingly, CCL20 induction is HDM specific as ragweed pollen and cockroach antigen do not induce CCL20 secretion and depend upon β-glucan recognition rather than protease activity (25). Although not much is known regarding the effects of Asp on dendritic cell recruitment in allergic settings, recent data indicate that CCR7 and its ligands CCL19 and CCL21, which are upregulated in asthma (36, 37), are involved in response to invasive aspergillosis (38). Interestingly, we show that both HDM and Asp induce significant recruitment of DCs to the draining lung lymph nodes. However, Asp induces greater DC accumulation, which is predominantly regulated by IL-13rα1. Thus, differential recruitment of dendritic cells in response to allergen challenge may determine the functional consequence of differential IL-4:IL-13 ratios in the lung and consequent eosinophilia (39). Directly related and supporting this hypothesis, we demonstrate that systemic sensitization of Il13ra1^−/− mice using Asp and alum and consequent local Asp challenge was capable of inducing IL-13Rα1-independent eosinophil lung accumulation. This result is consistent with a previous report that OVA- and alum-sensitized Il13ra1^−/− mice develop pulmonary eosinophilia (3). Yet, two differences were observed between these models: 1) in the OVA/alum model, lung eosinophilia was significantly decreased, whereas in the Asp/alum model eosinophil numbers in wild type and Il13ra1^−/− mice were similar (3) and 2) in response to OVA/alum, Il13ra1^−/− mice displayed a concomitant upregulation in neutrophil accumulation (3), whereas neutrophil levels remained similar to allergen-challenged wild type mice in response to Asp/alum. It is important to note that our overall findings are consistent with observations that STAT6-independent lung eosinophilia can occur following Asp (40). The finding that IL-13Rα1-independent eosinophilia can occur (as observed in the OVA/alum-sensitized mice (3) or in the lung tissue of Asp-challenged mice), identifies a pathway for eosinophil recruitment to the lung that appears to be primarily independent of classic eosinophil chemokines such as the eotaxins (15, 41, 42). A comprehensive summary of IL-13Rα1-dependent and independent pathways in response to the various experimental asthma models is shown in Table 1.

Table 1

Summary of IL-13Rα1-dependent and independent pathways in experimental asthma models

Various studies have shown IL-13Rα2-dependent TGF-β induction (43, 44). Our findings demonstrate that allergen-induced TGF-β production was completely dependent on IL-13Rα1. Similarly, TGF-β production in liver fibrosis following S. mansoni infection has been proposed to be independent of IL-13Rα1 (27). Despite this, we cannot exclude the possibility that IL-13:IL-13Rα1 interactions upregulate IL-13Rα2 expression, which mediates TGF-β production. Nonetheless, the finding that IL-13Rα1 is upstream of allergen-induced TGF-β production has significant implications for asthma related fibrosis. Although eosinophils may be a significant source for TGF-β expression in settings of allergic inflammation (45, 46), decreased allergen-induced TGF-β production is not likely due to eosinophil-derived TGF-β since TGF-β levels were abrogated even in the presence of eosinophilia (as in the HDM model). Nevertheless, it is still possible that IL-13Rα1 mediates TGF-β production in eosinophils and, therefore, that Il13ra1^−/− eosinophils may not be capable of producing TGF-β in the allergic lung.

Our results establish a specific and key role for IL-13 in driving the effector arm of allergic lung responses as allergen-induced IgE and Th2 cytokine production occurred independent of IL-13Rα1. Notably, while IL-13:IL-13Rα1 interactions are not involved in Th2 immune polarization in the lungs, they may have a role in Th2 polarization in mouse models of epicutaenous sensitization since Il13^−/−, Il4^−/− and Stat6^−/− mice display defective Th2 cytokine production in skin-draining lymph node cells following epicutaenous OVA sensitization (47).

In summary, our results establish the critical role for IL-13Rα1 in experimental asthma pathogenesis mediated by natural allergens following mucosal sensitization, conditions that may better mimic human asthma compared with experimental models that rely on intraperitoneal sensitization with adjuvants (e.g. alum). The finding that IL-13Rα1 regulates the key effector features of allergic asthma, independent of regulating adaptive immunity (as evidenced by sustained production of IgE and Th2 cytokines in Il13ra1^−/− mice) position IL-13Rα1 as a potent and promising target for asthma treatment. Furthermore, our data highlights that IL-13Ra1 mechanistically regulates aeroallergen-induced eosinophil recruitment by an extrinsic mechanism (likely dependent upon chemokine production) and aeroallergen-induced DC homing to draining lymph nodes. Finally, our results suggest that outcomes of future IL-13Rα1-targeted asthma therapy may vary in individuals according to the levels of allergen-induced IL-4.

Acknowledgments

We wish to thank Drs. Jamie Lee and Nancy Lee (Mayo Clinic, AZ) for the anti-MBP antibody and Shawna Hottinger for final edits to the manuscript.

Footnotes

Disclosures: A.M., E.T.C, D.S. and M.M. declare no conflict of interests. M.E.R. has an equity interest in reslizumab, a drug developed by Cephalon, Inc.

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