Background
Asthma exacerbations cause considerable morbidity and are frequently associated with rhinovirus and respiratory syncytial virus infections [
1,
2]. The two-hit hypothesis states that viral infections represent a second hit triggering acute asthma exacerbation in patients suffering from already established allergic lung inflammation as a first hit [
3]. There is evidence that viral RNAs cause exacerbation-associated inflammation and that dsRNA motifs (e.g. polyinosinic:polycytidylic acid (pIC)) trigger exacerbation similar to rhinovirus infections in models of experimental asthma [
4‐
6].
The IL-17 receptor family consists of five receptor subtypes (IL-17RA to IL-RE), which interact with different members of the IL-17 cytokine family (Il-17A to F) [
7,
8]. IL-17C is suggested to signal through a complex of IL-17RE and IL-17RA, whereas IL-17RA is also forming a heterodimeric receptor complex with IL-17RC for IL-17A signaling [
8]. IL-17RE is primarily expressed by epithelial cells and lymphocytes, such a Th17 cells, whereas IL-17RA is ubiquitously expressed [
9‐
13].
There is a functional overlap between IL-17A and IL-17C. Both cytokines mediate the expression of cytokines, chemokines, and antimicrobial peptides [
8]. However, IL-17A is expressed by immune cells (e.g. Th17 cells, tissue resident T cells), whereas IL-17C is mainly of epithelial origin [
8,
9,
12‐
14]. In vitro and in vivo studies showed that the expression of IL-17C in airway epithelial cells is induced by lung pathogens including rhinoviruses and that IL-17C promotes the recruitment of neutrophils into the lung [
12‐
22].
Studies suggest a function for IL-17A and IL-17RA in the development of allergic inflammation of the lung and airway hyper-responsiveness (AHR) [
5,
23‐
26]. It has been demonstrated that IL-17A promotes contractile force generation of airway smooth muscle through IL-17RA [
23,
24]. Because of the functional overlap between IL-17A and IL-17C and the corresponding receptor complexes IL-17RA/IL-17RC and IL-17RA/IL-17RE, we examined the function of IL-17RE in mouse models of OVA-induced experimental asthma and acute exacerbation thereof. We provide evidence that IL-17RE does not have a function in the development of allergic airway inflammation and AHR. However, our data indicate that IL-17RE contributes to pIC-triggered exacerbation once allergic airway inflammation has been established.
Material and methods
Mice
IL-17RE-deficient (Il-17re−/−) C57BL/6 mice were initially obtained from Mutant Mouse Resource and Research Center (MMRRC, USA). Female mice Il-17re−/− mice and their wild-type (WT) littermates were used at the age of 9–11 weeks. Breeding of animals and all animal experiments were approved by the Landesamt für Soziales, Gesundheit und Verbraucherschutz of the State of Saarland and by the animal ethics committee from the Department of State, Kiel, Germany. All experiments were done under consideration of the national guidelines for animal treatment.
Experimental protocol
WT and
Il-17re−/− mice were sensitized by i.p. injection with aluminum-hydroxide-adsorbed OVA (2 mg aluminum hydroxide (ThermoFisher, Waltham, USA) with 20 μg ovalbumin (Sigma-Aldrich, St. Louis, USA)) on days 1, 14, and 21. To induce acute allergic airway inflammation mice were exposed three times to an OVA aerosol (1% OVA in PBS) on days 26, 27, and 28. Control mice received PBS (i.p.) and were challenged with OVA aerosol. Mice were treated with pIC as described previously [
5]. In brief, mice were anaesthetized by i.p. injection of ketamine (105 mg/kg body weight, Bayer, Leverkusen, Germany) and xyalizine (7 mg/kg body weight, Serumwerk Bernburg AG, Bernburg, Germany) 2 h after the final OVA challenge. 100 μg pIC (Sigma-Aldrich, St. Louis, USA) dissolved in 20 μl sterile PBS or 20 μl PBS without pIC were administrated intranasally.
Bronchoalveolar lavage and cytokine measurements
Bronchoalveolar lavage (BAL) fluids were collected 24 h after the final OVA challenge as described before [
18,
21]. In brief, mice were euthanized, the tracheae were cannulated and BAL was performed with 1 ml of PBS flushed three times into the lungs. Numbers of immune cells were counted by using a hemocytometer (Innovatis AG, Reutlingen, Germany). Leukocytes were differentiated by DiffQuick Staining (Medion Diagnostics, Miami, FL, USA) on cytospin preparations. Lavaged lungs were homogenated in 1 ml PBS. BAL fluids and lung homogenates were centrifuged and the supernatants were kept at − 80 °C. Cytokines were measured by ELISA (R&D, Minneapolis, MN, USA).
Quantitative RT-PCR
RNA was isolated from lung tissue with Trizol Reagent (Life Technologies, USA) 4 h after treatment with pIC or PBS according to the manufacturer’s protocol. 2 μg of total RNA was used for the synthesis of cDNA and real-time PCR were performed as described before [
17,
18,
27,
28].
Lung histology
Immunohistochemical staining was done as described before [
17,
21]. In brief, lungs were fixed by instillation of 4% formalin buffered in PBS under a constant hydrostatic pressure (30 cm H
2O for 15 min), embedded in 1% agarose, cut into slices of exactly the same thickness, and embedded in paraffin. Samples were permeabilized with 0.5% Tween-20. Following primary antibodies were used: CD4 (ab183685, 1/100; Abcam, Cambridge, UK) and Ly6B (MCA771GA, 1/150; Bio-Rad, Munich, Germany). HRP-conjugated secondary antibodies (Histofine Simple Stain, Nichirei Biosciences Inc. Japan) were used. Randomly selected fields were evaluated for positive cells blinded to the investigator using the Visiopharm Integrator System (Visiopharm, Hoersholm, Denmark) on an Olympus BX51 microscope.
Lung function
Airway reactivity was assessed by methacholine (MCh, acetyl-b-methylcholine chloride; Sigma-Aldrich, St. Louis, MO, USA) provocation and recorded using invasive lung function assessment (FinePointe RC units; Data Sciences International, New Brighton, MN, USA) as described before [
5]. Mice were anesthetized with ketamine (90 mg/kg body weight; cp-pharma, Burgdorf, Germany) and xylazine (10 mg/kg; cp-pharma), cannulated, and mechanically ventilated with 150 ml/breath. Mice were provoked with increasing concentrations of MCh aerosols as indicated. Each aerosol provocation lasted for 30 s and was followed by 270s incubation time.
Statistical analysis
Comparisons were tested with two-way ANOVA (Tukey post-test) using the software Prism (GraphPad Software, San Diego, CA). The results were considered statistically significant for P < 0.05.
Discussion
In this study, we examined the function of the IL-17 receptor IL-17RE in pIC-triggered exacerbation of experimental asthma. The main findings are: (1) IL-17RE is not required for the development of OVA-induced lung inflammation and AHR; (2) IL-17RE does not mediate pIC-induced pulmonary inflammation in the absence of allergic inflammation; (3) IL-17RE promotes pIC-triggered inflammation once allergic airway inflammation has established.
Preclinical studies showed that IL-17A and IL-17RA mediate the development of allergic inflammation of the airways and AHR through the recruitment of inflammatory cells and the induction of smooth muscle contraction (22–25). As it has been shown that IL-17A and IL-17C both require IL-17RA for the induction of inflammatory mediators in target cells and IL-17C promotes Th17 cell responses [
11,
33,
34] we hypothesized that the specific IL-17C receptor IL-17RE has a function in OVA-induced experimental asthma. However, we did not detect any significant difference in the amounts of pulmonary cytokines, numbers of pulmonary immune cells, and AHR between WT mice and
Il-17re−/− littermates in the presence or absence of allergic lung inflammation. Therefore, our data do not indicate that IL-17RE has a role in the development of OVA-induced allergic lung inflammation and AHR. This finding can be explained by the relatively low expression of the IL-17RE ligand IL-17C in the absence of additional stimuli, such as ligands for pattern recognition receptors, the suppressive effect of Th2 cytokines on IL-17C expression, and a possible lack of IL-17RE expression in airway smooth muscles [
12,
16,
35].
As intranasal inoculation with pIC mimics viral infections we studied the function of IL-17RE in pIC-triggered lung inflammation [
4‐
6]. In our experimental setup, intranasal application of pIC resulted in a moderate lung inflammation, which was slightly, but not significantly decreased in
Il-17re−/− mice. However, once allergic inflammation had been established, pIC-triggered lung inflammation in an IL-17RE-depended manner. Pulmonary concentrations of inflammatory cytokines, numbers of neutrophils in BAL fluids, and numbers of Ly6B
+ in the lung parenchyma were decreased in
Il-17re−/− mice. Thus, IL-17RE promotes inflammation in an already diseased lung.
There is evidence that IL-17RE meditates T cell activation, including the expression of effector cytokines (e.g. IL-17A) [
11,
33,
36]. It has been shown that IL-17RE is highly expressed in Th17 cells and that the IL-17C/IL-17RE-axis enhances the expression of cytokine by effector Th17 cells in a in a model of autoimmune disease [
11]. IL-17RE is also highly expressed by liver resident CD4
+ T cells and natural killer T cells and augments T cell function in autoimmune hepatitis together with IL-17C [
37]. Deficiency of IL-17RE also provided protection in a model of crescentic nephrotoxic nephritis, which was associated with a reduced Th17 response [
33]. Thus, increased expression of inflammatory mediators by CD4
+ and natural killer T cells already present in asthmatic mice could be a mechanism by which IL-17RE increases pulmonary inflammation in pIC-treated mice.
Ablation of IL-17RE resulted in a partial reduction of AHR in pIC-treated mice. It has been shown that neutrophil depletion partially decreases AHR in experimental asthma [
23]. In addition, Toussaint et al. showed that DNA released by neutrophils promotes rhinovirus-induced type-2 allergic asthma exacerbation [
38]. Thus, in our experimental setup, pulmonary neutrophils may affect MCh-induced AHR, whereas IL-17RE does not seem to have a direct function in the induction of smooth muscle contraction.
Conclusion
In summary, our findings suggest that IL-17RE does not have a function in the development of allergic lung disease. However, once allergic inflammation has been established, IL-17RE mediates virus-triggered lung inflammation.
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