Viral infection of the respiratory tract represents the major cause of acute asthma exacerbations. dsRNA is produced as an intermediate during replication of respiratory viruses and triggers immune responses via TLR3. This study aimed at clarifying the mechanisms underlying TLR3 triggered exacerbation of experimental allergic asthma. The TLR3 ligand poly(inosinic-cytidylic) acid was applied intranasally to mice with already established experimental allergic asthma. Airway inflammation, cytokine expression, mucus production, and airway reactivity was assessed in wild-type, IL-17A, or IL-23p19–deficient, and in NK cell–depleted mice. Local application of poly(inosinic-cytidylic) acid exacerbated experimental allergic asthma in mice as characterized by enhanced release of proinflammatory cytokines, aggravated airway inflammation, and increased mucus production together with pronounced airway hyperresponsiveness. This was further associated with augmented production of IL-17 by Th17 cells and NK cells. Whereas experimental exacerbation could be induced in IL-23p19–deficient mice lacking mature, proinflammatory Th17 cells, this was not possible in mice lacking IL-17A or in NK cell–depleted animals. These experiments indicate a central role for IL-17 derived from NK cells but not from Th17 cells in the pathogenesis of virus-triggered exacerbation of experimental asthma.
Exacerbations represent a distinct and clinically important characteristic of asthma imposing considerable morbidity on patients and constituting a major economic burden on health care systems. Asthma exacerbations are defined as acute episodes of progressive worsening, shortness of breath, cough, wheezing, and chest tightness or some combination of these symptoms (1).
The inflammatory infiltrate of acute asthma exacerbations displays a heterogeneous pattern with large numbers of both, eosinophils and neutrophils, which is different from the pattern seen in chronic disease (2). Especially in exacerbated severe asthma, airway inflammation is further associated with markedly increased levels of IL-8 and proinflammatory mediators such as IL-6 and TNF (3). These observations suggest that the pathogenesis of acute asthma exacerbations is different from that seen in chronic disease.
Epidemiological surveys suggested viral infection of the upper respiratory tract as the main trigger of acute asthma exacerbations based on a positive correlation between exacerbations and symptomatic colds (4). Thus, in 44% of adults and 80–85% of school-age children with acute asthma exacerbations, viral infection of the respiratory tract could be detected (5). According to the “two-hit hypothesis,” such viral infections represent the “second hit” triggering acute asthma exacerbation in patients that already experienced allergen sensitization and subsequent asthma development as a “first hit” (6).
Rhinoviruses and respiratory syncytial virus are by far the most frequently detected virus types that cause asthma exacerbations (7). Other respiratory viruses associated with asthma exacerbation include influenza viruses, human metapneumovirus, coronaviruses, and parainfluenzaviruses (5). Remarkably, all these viruses use ssRNA to encode their genome, which displays dsRNA motifs in its tertiary structure or as an intermediate during viral replication. In turn, such dsRNA motifs can be sensed by the immune system via TLR3 or retinoic acid–inducible gene-I (8, 9), suggesting a central role of this receptors in the pathogenesis of acute asthma exacerbations. This hypothesis is supported by mouse experiments demonstrating that not only rhinovirus infection but also local application of viral dsRNA or poly(inosinic-cytidylic) acid (pIC) alone triggers exacerbation of experimental asthma in mice (10–12). However, the underlying mechanisms of this disease pattern remain completely unclear. Therefore, the aim of this study was to identify the cells and mediators directing the immune response ultimately leading to acute exacerbation of experimental asthma.
Materials and Methods
Female, 6- to 8-wk-old C57BL/6 (Charles River Laboratories, Sulzfeld, Germany), BALB/c, IL-17A–deficient (−/−) (13), and IL-23p19−/− mice (14) on C57BL/6 genetic background were housed under specific pathogen free conditions. They received OVA-free diet and water ad libitum. All experiments were in accordance with the German Animal Protection Law and were approved by the Animal Research Ethics Board of the Ministry of Environment (Kiel, Germany).
Animal treatment protocol
Mice were sensitized to OVA by three i.p. injections of 10 μg OVA (OVA grade VI; Sigma-Aldrich, Deisenhofen, Germany) adsorbed to 150 μg aluminum hydroxide (imject alum; Thermo, Rockford, IL) on days 1, 14, and 21. To induce acute allergic airway inflammation, mice were exposed three times to an OVA (OVA grade V; Sigma-Aldrich) aerosol (1% w/v in PBS) on days 26, 27, and 28 (15). Intranasal application of the TLR3 ligand pIC (Sigma-Aldrich) was performed after allergic airway inflammation had already been established on day 28. Therefore, mice, anaesthesized with ketamin and xylazin, received 200 μg pIC dissolved in 50 μl sterile PBS (OVA + pIC group) or only 50 μl PBS as a control (OVA group) intranasally 1 h after the last OVA aerosol challenge. For NK cell depletion, on day 27, wild-type C57BL/6 mice received i.p. injection of 40 μl anti–Asialo GM (monosialotetrahexosylganglioside) 1 polyclonal antiserum (Wako Chemicals, Richmond, VA) (16) that was reconstituted in sterile 0.9% NaCl solution as recommended by the manufacturer. NK cell depletion efficacy was determined by flow cytometric analysis using staining against CD-3 and NK1.1 of the spleen cells of treated mice as described below and achieved >85%. All animals were sacrificed by cervical dislocation under deep anesthesia on day 29. Negative control animals were sham sensitized to PBS and subsequently challenged with OVA aerosol (PBS group). Eight animals per group were used, if not stated otherwise.
Lungs were rinsed with 1 ml fresh, ice-cold PBS containing protease inhibitor (Complete, Roche, Basel, Switzerland) via a tracheal canula, and obtained cells were counted using a Countess automated cell counter (Life Technologies, Darmstadt, Germany). Fifty microliter aliquots of lavage fluids were cytospined and stained with Diff-Quick (DADE Diagnostics, Unterschleissheim, Germany), and cells were microscopically differentiated, according to morphologic criteria (15).
Lungs were fixed ex situ with 4% (w/v) paraformaldehyde via the trachea under constant pressure, removed, and stored in 4% paraformaldehyde. Lung tissues were embedded in paraffin. For analysis of lung inflammation, 2-μm sections were stained with periodic acid–Schiff (PAS) or with H&E, respectively. Photomicrographs were recorded by a digital camera (DP-25; Olympus, Tokyo, Japan) attached to a microscope (BX-51; Olympus) at 40- and 100-fold magnification using Olympus cell^A software. For mucus assessment, systematic uniform random samples of lung tissue were taken according to standard methods (17) including the orientator technique (18). The surface area of mucin-containing goblet cells (Sgc) per total surface area of airway epithelial basal membrane (Sep) and the volume of PAS-stained epithelial mucin (Vmucin) per Sep were determined using a computer-assisted stereology tool box (newCAST, Visiopharm, Hoersholm, Denmark) (19, 20), according to the following formulas: Sge/Sep = ∑Igc/∑Iep and Vmucin/Sep = LP × (∑Pmucin/2) × ∑Iep, where ∑Igc is the sum of intersections of test lines with goblet cells, ∑Iep is the sum of all intersections of test lines with the epithelial basal membrane, ∑Pmucin is the sum of all points hitting mucin, and LP is the test-line length at final magnification.
Assessment of airway responsiveness to methacholine
Airway reactivity was assessed by methacholine (MCh) provocation testing, whereas respiratory mechanics were recorded using invasive lung function assessment (FinePointe RC units; Data Sciences International, New Brighton, MN). Mice were anesthetized with ketamine (90 mg/kg body weight [BW]; cp-pharma, Burgdorf, Germany) and xylazine (10 mg/kg BW; cp-pharma), tracheotomized with a cannula, and mechanically ventilated with 150 μl/breath. The neuromuscular activity was blocked with pancuronium bromide (0.5 mg/kg BW; Sigma-Aldrich). Mice were allowed to stabilize for 5 min before measurements started. MCh (acetyl-β-methylcholine chloride; Sigma-Aldrich) provocation testing started with PBS, followed by MCh aerosols with increasing concentrations (0, 3.125, 6.25, 12.5, 25, 50, and 100 mg/ml). Each aerosol provocation lasted for 30 s, followed by 270-s incubation time. Airflow and transpulmonary pressure in response to MCh inhalation were recorded and analyzed with FinePointe Review software (version 184.108.40.206; Data Sciences International), which calculated airway resistance (cm H2O/ml/s). After measurement terbutaline hemisulfat (9 μg/kg BW; Sigma-Aldrich) was applied i.m. to relax airway smooth muscle cells allowing further analysis of lung tissues.
Assessment of bronchoalveolar lavage cytokines
Preparation of single-cell suspensions from lungs
For Ag-specific restimulation and flow cytometric analysis, single-cell suspensions of lungs were prepared from mice on day 29. Mice were anesthetized and injected i.p. with 150 U heparin (Ratiopharm, Ulm, Germany). Lungs were perfused through the right ventricle with warm PBS. Once lungs appeared white, they were removed and sectioned. Dissected lung tissue was then incubated with collagenase A (0.7 mg/ml; Roche Diagnostics, Mannheim, Germany) and DNase (30 μg/ml; Sigma-Aldrich) at 37°C for 2 h. Digested lung tissue was gently disrupted by subsequent passage through a 100-μm pore size nylon cell strainer. Recovered vital lung cells were counted using an Countess Automated Cell Counter (Life Technologies, Darmstadt, Germany), diluted in complete IMDM (Life Technologies), supplemented with 10% FCS (Life Technologies), 0.05 mM 2-ME (Sigma-Aldrich, Deisenhofen, Germany), and penicillin and streptomycin (100 U/ml and 100 μg/ml; Life Technologies) and used for further experiments.
Intracellular cytokine staining and flow cytometric analysis
For detection of intracellular IL-17A, an intracellular cytokine staining kit was used (BD Biosciences). Briefly, single-cell suspensions were prepared at day 29 of the experimental asthma protocol and 1.8 × 106
Results are presented as mean values ± SEM. One-way ANOVA with subsequent Tukey’s test was used to determine the significance of differences between animal groups.
Local application of pIC exacerbates experimental allergic asthma
A well-established mouse model of experimental allergic asthma (21, 22) was used as a basis to trigger acute asthma exacerbation. As expected, this protocol resulted in goblet cell metaplasia, mucus hyperproduction, allergic airway inflammation, and airway hyperresponsiveness (AHR) (Fig. 1).
To mimic a “second hit” according to the “two hit hypothesis” (6), pIC, a familiar surrogate for dsRNA capable of activating TLR3 and retinoic acid–inducible gene-I (9, 23), was applied intranasally 49 h after the first local allergen contact and, thus, at a time point when experimental allergic asthma had already been established. Compared with mice with experimental asthma alone, this “second hit” induced an increase in goblet cell metaplasia (Fig. 1A) and mucus production as evident by significantly augmented epithelial basement membrane surface area covered by mucus and stored mucus volume per epithelial basement membrane surface area (Fig. 1B). Furthermore, pIC treatment aggravated allergic airway inflammation (Fig. 1C) with significantly increased numbers of both, neutrophils and eosinophils, in bronchoalveolar lavage (BAL) fluid (Fig. 1D). Compared with animals with experimental allergic asthma, pIC-treated animals revealed a considerable increase in reactivity toward inhaled MCh (Fig. 1E). However, because of a relatively high SD, this increase does not reach the level of significance. Thus, local application of pIC resulted in acute exacerbation of experimental asthma as evident by aggravation of all typical disease hallmarks.
pIC-triggered exacerbation of experimental asthma is associated with increased cytokine and chemokine production
To characterize the inflammatory response underlying pIC-triggered exacerbation of experimental asthma, the expression of cytokines and chemokines was analyzed by quantitative RT-PCR. In total lung tissue homogenates, pIC-treated animals revealed highest expression levels of numerous mediators including typical Th2 type cytokines (IL-4, IL-5, IL-9, and IL-13) (Fig. 2A–D), generally proinflammatory cytokines (IL-6 and TNF) (Fig. 2I, 2J), cytokines associated with Th17 immune responses (IL-17A and IL-23p19) (Fig. 2K, 2L) and neutrophilotactic chemokines (KC, RANTES, and IP-10) (Fig. 2 F–H). Interestingly, the expression of the Th1 type cytokine IFN-γ remained unchanged (Fig. 2E). A similar profile could also be detected in BAL fluid, where the levels of IL-1β, IL-4, IL-5, IL-6, IL-17A, TNF, and KC were highest in mice with exacerbated experimental asthma (Table I).
pIC-triggered exacerbation of experimental asthma depends on IL-17A
IL-17A has been associated with neutrophil infiltration into asthmatic airways, and its expression was reported to correlate with disease severity in both human patients and mice with experimental allergic asthma (24, 25). Because the expression of IL-17A was enhanced in mice with exacerbated experimental asthma, we hypothesized that the development of pIC-triggered exacerbation of experimental asthma is mediated by IL-17. To test this hypothesis, we applied the protocols for the induction and exacerbation of experimental asthma detailed above to IL-17A−/− mice. OVA sensitization and subsequent OVA aerosol challenge triggered goblet cell metaplasia (Fig. 3A), increased mucus production (Fig. 3B), and allergic airway inflammation (Fig. 3C, 3D) comparable to those seen in wild-type animals. Remarkably, intranasal application of pIC that triggered exacerbation of experimental asthma in wild-type mice did not result in any significant alteration of mucus production, airway inflammation, and AHR in IL-17A−/− mice. These data suggest a central role for IL-17A in mediating pIC-triggered exacerbation of experimental asthma in mice.
Numbers of IL-17A producing Th17 and NK cells are enhanced in pIC-triggered exacerbation of experimental asthma
Thus, we aimed at identifying the cellular source of IL-17A in these animals. For this purpose, leukocytes were isolated from lungs, restimulated in vitro (with PMA and Ionomycin), stained for intracellular IL-17A, and subsequently analyzed by flow cytometric measurement. Compared with healthy controls, animals with experimental asthma revealed slightly enhanced cell numbers of IL-17A+/CD4+/CD3+ Th17 (Fig. 4A, 4B) and of IL-17A+/CD3+NK1.1+/CD1d-αGalCer− NK cells (Fig. 4C, 4D). However, the percentages of both, Th17 cells and IL-17A–producing NK cells, were markedly increased in mice with exacerbated experimental asthma (Fig. 4E). Although invariant NKT cells have been described previously to produce IL-17A in response to pIC stimulation (26), we did not observe any IL-17A+NK1.1+CD1d-aGalCer+ NKT cells in any of the groups.
pIC-triggered exacerbation of experimental asthma is independent of IL-23
To test the hypothesis that Th17 cells are the major source for IL-17A in pIC-triggered exacerbation of experimental asthma, IL-23p19−/− mice that are still able to produce IL-17A but cannot generate mature IL-17A–producing Th17 cells (27, 28) were used. Animals sensitized to OVA and challenged with OVA aerosol displayed a phenotype of experimental asthma comparable to that of wild-type animals by points of goblet cell metaplasia, mucus production, and airway inflammation; however, induction of AHR was not possible by using this protocol (Fig. 5). In contrast to our hypothesis, intranasal application of pIC resulted in a significant increase of goblet cell metaplasia, mucus production (Fig. 5A, 5B), airway inflammation (Fig. 5C), and numbers of neutrophils and eosinophils in BAL (Fig. 5D) as compared with asthma controls. Assessment of IL-17A in BAL fluid revealed amounts at the same level as observed in wild-type mice with highest amounts in pIC-treated animals with experimental asthma (Fig. 5E).
IL-17A producing NK cells play a critical role in pIC-triggered exacerbation of experimental asthma
Consequently, we hypothesized that NK cells, the second leukocyte subpopulation that displayed increased production of IL-17 in this setting, are the major source of IL-17 in pIC-triggered exacerbation of experimental asthma. Thus, wild-type mice were subjected to the protocol for the induction of exacerbated experimental asthma and were additionally treated with an anti–Asialo GM1 polyclonal antiserum, which is a standard method to deplete NK cells (16). This NK cell depletion led to a reduced overall response compared with untreated mice undergoing the induction of experimental asthma (Fig. 1).
Interestingly, this treatment had no significant effect on the production of neutrophilotactic mediators (IL-6, KC, MCP-1, RANTES, and TNF) and accordingly on neutrophil infiltration into the bronchoalveolar lumen. However, NK cell–depleted mice did not display any significant increase in goblet cell metaplasia, mucus production, and infiltration of eosinophils into the BAL as compared with asthma controls (Fig. 6A–D). NK cell depletion also inhibited an increase in IL-17 production as assessed in BAL fluid (Fig. 6E).
We used intranasal application of pIC, a familiar surrogate of dsRNA, as a second hit to exacerbate the phenotype of a well-established mouse model of experimental allergic asthma by points of mucus production, airway inflammation, and AHR. In contrast to infection with rhinovirus or respiratory syncytial virus, this strategy successfully aggravated all hallmarks of the disease and was not restrained by viral host specificity or inability to replicate in murine tissues (29, 30). Exacerbation of experimental asthma was associated with increased expression and production of a plethora of proinflammatory mediators including Th2 type cytokines, Th17 type cytokines, and neutrophilotactic chemokines. Mechanistically, we could show that among this multitude of mediators IL-17A, which in our model is mainly produced by NK cells and Th17 cells, is essential for the induction of such an asthma exacerbation because intranasal application of pIC did not exacerbate experimental asthma in IL-17A−/− mice. In contrast, in IL-23p19−/− mice, which are devoid of mature, proinflammatory Th17 cells, and IL-17A–producing γδ T cells, local pIC application caused asthma exacerbation. Taken together with the finding that most hallmarks of pIC-triggered experimental asthma exacerbation are lacking in NK cell–depleted mice, we suggest NK cells rather than Th17 cells as main producers of IL-17A and, thus, of central importance in this setting.
In mice with exacerbated experimental asthma, we observed an augmented influx of eosinophils, typical effector cells of an allergic immune response, as well as of neutrophils, typical effector cells of antimicrobial immune responses. The recruitment of these two effector cell types is differentially regulated. Because IL-5 is the major survival factor for peripheral eosinophils, the enhanced expression of IL-5 and the eosinophilotactic chemokine RANTES can explain the increased appearance of eosinophils (31). Correspondingly, in mice the recruitment of neutrophils to the airways is mainly regulated by KC, TNF, and IL-6 (32), which were also highly expressed in exacerbated experimental asthma.
We also observed increased expression of IL-17A, which lead us to investigate its role in exacerbating experimental asthma. Mice deficient for this cytokine developed a typical Th2-dominated experimental asthma as characterized by mucus hyperproduction, allergic airway inflammation, and development of AHR. However, intranasal application of pIC did not aggravate any of these disease hallmarks and, thus, failed to exacerbate experimental asthma in the absence of IL-17A.
Actually, the role of IL-17A in the pathogenesis of human asthma as well as in experimentally induced asthma is discussed controversially. Whether or not IL-17A contributes to the development of experimental asthma seems to depend, for example, on the protocol used to induce the respective asthma phenotype. On the one hand, in adjuvant-free mouse models, IL-17A seems to be required for the sensitization against an allergen and the initiation of Ag-mediated airway inflammation (25, 33). On the other hand, IL-17A deficiency had no significant effect on sensitization or the phenotype of experimental asthma, if mice were sensitized systemically by using an adjuvant (34). The results of our experiments are in line with that latter study and display that IL-17A is not required for the development of experimental allergic asthma.
The observation that the “second hit”—namely the local application pIC—did not lead to acute exacerbation of the already established Th2-dominated asthma phenotype in IL-17−/− mice strongly indicates a critical role for this cytokine within the underlying pathogenetic mechanisms. Indeed, IL-17 has the properties to aggravate each of the hallmarks of experimental asthma: mucus hyperproduction, airway inflammation, and AHR. IL-17A directly and indirectly stimulates the expression of the mucin genes MUC5AC and MUC5B in human as well as murine airway epithelial cells (35). It further enhances airway smooth muscle cell contraction and promotes the development of AHR (36, 37). In addition, IL-17A was described to affect airway inflammation through several pathways. IL-17A induces indirect activation and infiltration of neutrophils into the airways, partially through induction of KC expression in structural cells of the airways (38, 39). Furthermore, IL-17A seems also to be able to amplify Th2 immune responses (40, 41). Each of these IL-17A properties can explain why intranasal administration of pIC leads to exacerbation of experimental asthma in wild-type animals but not in mice lacking IL-17.
In wild-type mice with exacerbated experimental asthma, not only the expression of IL-17 but also of IL-23p19 was significantly enhanced. Furthermore, the numbers of Th17 cells were markedly increased. Because IL-23 is essential for the full and sustained differentiation of Th17 cells and IL-23p19−/− mice are not able to generate active effector Th17 cells (27), we hypothesized that IL-23p19−/− mice would be insensitive to pIC-triggered exacerbation of experimental asthma. Remarkably, deficiency in IL-23p19 in contrast to IL-17A deficiency did not block the development of pIC-triggered asthma exacerbation. Increases in goblet cell metaplasia, mucus production, and airway inflammation were equivalent to the effects seen in wild-type mice. Alike IL-17A, the role of IL-23 in asthma pathogenesis is discussed controversially. IL-23 expression is elevated in mice with experimental asthma and exogenous administration or local overexpression of IL-23 resulted in aggravation of the disease (42, 43). In contrast, lack of IL-23 had no effect on the development of experimental asthma in mice (34). The latter study is in line with our results, whereas IL-23p19−/− animals developed a disease phenotype that was absolutely comparable to wild-type mice with the exception of AHR. However, although IL-23p19 expression was increased in wild-type mice with exacerbated experimental asthma, local administration of pIC nonetheless lead to an aggravated disease phenotype in IL-23−/− mice. These results indicate that IL-23 is not essential for pIC-triggered exacerbation of experimental asthma.
Because IL-23 is required to generate mature, active Th17 cells or IL-17–producing γδ T cells, IL-23p19−/− mice do not display these cells (27). Thus, our experiments further indicate that such cells are not essential for IL-17A–dependent exacerbation of experimental asthma. However, several other immune cells have been identified to produce IL-17A including Th2 cells (41), macrophages (44), mast cells (45), neutrophils (46), and NK cells (47). In our setting, mice with pIC-triggered exacerbation of experimental asthma displayed markedly increased numbers of IL-17–producing NK cells.
Accordingly, IL-17A levels in BAL were significantly lower in NK cell–depleted mice. As deduced from the experiments using IL-17A−/− mice, it was expectable that local application of pIC would have no effect on the production of neutrophilotactic mediators and, consequently, on neutrophil numbers in BAL. However, levels of neutrophilotactic mediators such as KC, IL-6 or TNF remained nearly untouched by the anti–Asialo treatment, which could explain that enhanced infiltration of neutrophils into the bronchoalveolar lumen is still present in NK cell–depleted animals. On the one hand, it is accepted that NK cells together with neutrophils can act as potent antiviral effectors (48, 49) so that depletion of one effector cell population—namely the NK cells—could lead to a compensatory effect to maintain an effective antiviral defense. On the other hand, it has been demonstrated that depletion of NK cells in vivo could lead in enhanced neutrophil infiltration into inflamed tissue, if NK cells exert regulatory functions (50).
Nevertheless, in NK cell–depleted mice local application of pIC did not result in increased goblet cell hyperplasia, mucus production, or infiltration of eosinophils. Thus, except from more neutrophils in the BAL, all other characteristics of experimental asthma exacerbation are missing when NK cells are absent. These data strongly suggest a critical role of IL-17A–producing NK cells to amplify the already established Th2 cell response and, thus, to aggravate hallmarks of experimental asthma in mice. The induction of IL-17 production in such NK cells can be induced independently from IL-23 by IL-6 (47), which could explain our findings in IL-23p19−/− mice and is in line with a markedly increased production of IL-6 in wild-type mice with pIC-triggered asthma exacerbation. Among other cells of the innate immune system NK cells have been suggested as an early source of IL-17A in response to viral or bacterial infection that act before a Th17 cell response is initiated (51). Albeit such a rapid IL-17A response represents an effective first-line defense mechanism against invading pathogens including viruses, in case of bronchial asthma, it could deploy pathologic side effects by amplifying the already established Th2 type immune response and, thus, leading to acute exacerbation.
In summary, we have shown that 1) pIC triggered exacerbation is mediated by IL-17A and 2) that the increase in IL-17A is not mediated by IL-23 but NK cells. Taken together, this study provides new mechanistic insights into the inflammatory responses underlying viral-induced asthma exacerbation suggesting TLR3 and/or IL-17A–producing NK cells as potential targets for new therapeutic approaches.
The authors have no financial conflicts of interest.
We acknowledge the National Institutes of Health Tetramer Core Facility (Contract HHSN272201300006C) for the provision of brilliant violet 421-labeled mCD1d/PBS57 tetramers. We thank Juliane Artelt, Franziska Beyersdorf, and Linda Lang for excellent technical assistance.
This work was supported by German Center for Lung Research Grant DZL AA2.1 and Deutsche Forschungsgemeinschaft Cluster of Excellence Grant EXC306.
Abbreviations used in this article:
- airway hyperresponsiveness
- bronchoalveolar lavage
- body weight
- keratinocyte chemoattractant
- periodic acid–Schiff
- poly(inosinic-cytidylic) acid.
- Received October 2, 2014.
- Accepted April 11, 2015.
- Copyright © 2015 by The American Association of Immunologists, Inc.