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Original Research: Asthma |

Toll-like Receptor 3 Stimulation Causes Corticosteroid-Refractory Airway Neutrophilia and Hyperresponsiveness in MicePoly(I:C)-Induced Airway Neutrophilia FREE TO VIEW

Genki Kimura, PhD; Keitaro Ueda, MSc; Shouichi Eto, MSc; Yuji Watanabe, MSc; Takashi Masuko, PhD; Tadashi Kusama, PhD; Peter J. Barnes, MD, FCCP; Kazuhiro Ito, DVM, PhD; Yasuo Kizawa, PhD
Author and Funding Information

From the Department of Physiology and Anatomy (Drs Kimura, Masuko, Kusama, and Kizawa and Messrs Ueda, Eto, and Watanabe), Nihon University School of Pharmacy, Funabashi, Chiba, Japan; and Airway Disease Section (Drs Barnes and Ito), National Heart and Lung Institute, Imperial College, London, England.

Correspondence to: Yasuo Kizawa, PhD, Department of Physiology and Anatomy, Nihon University School of Pharmacy, 7-7-1, Narashinodai, Funabashi, Chiba 274-8555, Japan; e-mail: kizawa.yasuo@nihon-u.ac.jp


Funding/Support: This research was funded by “High-Tech Research Center” Project for Private Universities (2007-2011): matching fund subsidy from MEXT (Japan; to Dr Kizawa).

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details.


Chest. 2013;144(1):99-105. doi:10.1378/chest.12-2610
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Background:  RNA virus infections, such as rhinovirus and respiratory syncytial virus, induce exacerbations in patients with COPD and asthma, and the inflammation is corticosteroid refractory. The main aim of this study is to establish a murine model induced by a Toll-like receptor 3 (TLR3) agonist, an RNA virus mimic, and investigate the response to corticosteroid.

Methods:  A/J mice were given polyinosinic-polycytidylic acid (poly[I:C]), a TLR3 agonist, intranasally, in the presence or absence of cigarette smoke exposure. Inflammatory cell accumulation and C-X-C motif chemokine (CXCL) 1, interferon (IFN), and CXCL10 production in BAL fluid (BALF) were determined by flow cytometry and enzyme-linked immunosorbent assay, respectively, and airway hyperresponsiveness (AHR) to histamine/methacholine was determined by a two-chambered, double-flow plethysmography system. BALB/c and C57BL/6J mice were also used for comparisons.

Results:  Intranasal treatment of poly(I:C) significantly induced airway neutrophilia; production of CXCL1, IFN-β, and CXCL10; and necrotic cell accumulation in BALF. It also increased airway responsiveness to histamine or methacholine inhalation. This poly(I:C)-dependent airway inflammation and AHR was not inhibited by the corticosteroid fluticasone propionate (FP) (up to 0.5 mg/mL intranasal), although FP strongly inhibited lipopolysaccharide (TLR4 agonist)-induced airway neutrophilia. Furthermore, cigarette smoke exposure significantly increased TLR3 expression in murine lung tissue and exacerbated poly(I:C)-induced neutrophilia and AHR.

Conclusions:  These results suggest that TLR3 stimulation is involved in corticosteroid-refractory airway inflammation in lung, which is enhanced by cigarette smoking, and this may provide a model for understanding virus-induced exacerbations in COPD and their therapy.

Figures in this Article

Respiratory RNA virus infections, such as human rhinovirus (HRV), respiratory syncytial virus (RSV), and influenza virus, are common causes of exacerbations of COPD and asthma1,2 and are associated with increased symptoms and reduced lung function, leading to substantial morbidity and mortality. At a cellular level, HRV, RSV, and influenza virus induce several neutrophil chemoattractants, such as C-X-C motif chemokine (CXCL) 8 (IL-8) in human epithelial cells or sputum obtained from subjects challenged with virus or murine CXCL1 (KC) in BAL fluid (BALF) in mice.38

Virus-induced lung inflammation is known to be corticosteroid insensitive. For example, symptoms after HRV challenge were not improved or even worsened by prednisolone,9 and a corticosteroid enhanced inflammation in RSV-infected ovalbumin-sensitized allergic mice.10 However the molecular mechanisms of corticosteroid insensitivity by virus infection have not been clarified. Several molecular mechanisms of corticosteroid insensitivity seen in COPD and asthma have been elucidated at least, including overexpression of transcription factors, reduced histone deacetylase-2 activity, increased decoy receptors, and posttranslational modifications of the glucocorticoid receptor.11

The double-stranded RNA (dsRNA) generated during RNA virus infection stimulates Toll-like receptor 3 (TLR3), which is expressed in immune cells such as macrophages, natural killer cells, and nonimmune cells, such as airway smooth muscle cells and epithelial and endothelial cells.12 The receptor activates nuclear factor-κB to induce an inflammatory response13,14 as well as inducing antiviral mechanisms via activation of an interferon (IFN) regulatory factor 3. Thus, many reports support that polyinosinic-polycytidylic acid (poly[I:C]) stimulation mimics several aspects of virus-dependent inflammation. In the present study, to clarify the mechanisms for exacerbations of COPD and asthma due to viral infection, we investigated the contribution of TLR3 signaling on airway neutrophilia and airway hyperresponsiveness (AHR) in naive or cigarette smoke-exposed mice and investigated the effects of corticosteroids on this poly(I:C)-induced airway neutrophilia and AHR.

Drug and Chemicals

The materials used were obtained from the following sources: fluticasone propionate (FP), dexamethasone, anti-β-actin (AC-15) antibody, and propidium iodide (Sigma-Aldrich Co, LLC); HRP-conjugated anti-rabbit IgG and anti-mouse IgG and ECL Plus (GE Healthcare Life Sciences); fluorescein isothiocyanate (FITC)-conjugated antimacrophage (MOMA2) antibody and antineutrophils (7/4) antibody (Acris Antibodies GmbH); DC protein assay kit (BioRad Laboratories, Inc); poly(I:C) (low molecular weight) (InvivoGen, San Diego, CA); and anti-TLR3 antibody (Abcam plc).

Animals

Specific pathogen-free A/J, BALB/c, and C57BL/6J mice (male, 5 weeks old) were purchased from Sankyo Labo Service Co Inc and adapted for 1 week in a temperature (24° ± 1°C) and humidity (55% ± 5%) controlled room with a 12-h day-night cycle. The mice were reared on a standard diet and tap water ad libitum. All animal studies were performed in accordance with the guidelines of the Nihon University Animal Care and Use Committee.

Poly(I:C) and Drug Administration

Poly(I:C) (1 mg/mL) was administered intranasally bid for 1 to 3 days under anesthesia with 3% isoflurane. FP was dissolved in 2% dimethyl sulfoxide in saline and administered intranasally in volume of 50 μL at 2 h before each poly(I:C) dosing, or FP was treated at 24 h before the first poly(I:C) administration in addition to at 2 h before every poly(I:C) dosing in some experiments (Fig 1).

Figure Jump LinkFigure 1. Effect of a corticosteroid on poly(I:C) induced airway neutrophilia and CXCL1 production in A/J mice. Poly(I:C) (1.0 mg/mL) was administered intranasally bid for 1 to 3 days. A, Effect of FP (0.005, 0.05, and 0.5 mg/mL) on BAL neutrophil numbers. B, Effect of FP (0.005, 0.05, and 0.5 mg/mL) on BAL CXCL1 concentrations. C, Effect of FP (0.005, 0.05, and 0.5 mg/mL) on BAL necrotic cells. Each value is presented as mean ± SEM (n = 4-7). Significant difference from time-matched control: #P < .05 or ##P < .01. FP (0.005, 0.05, and 0.5 mg/mL) was treated at 24 h before the first poly(I:C) administration in addition to at 2 h before every poly(I:C) dosing showing effects on BAL neutrophils (A), and CXCL1 (B). Each value is presented as mean ± SEM (n = 5-6). D, LPS (0.1 mg/mL) was administered intranasally 2 h after FP dosing (0.005, 0.05 and 0.5 mg/mL) and BAL fluid collected 3 h after LPS dosing. Each value is presented as mean ± SEM (n = 5). Significant difference from control: #P < .05, ##P < .01, or ###P < .001, and from LPS: ***P < .001. Con = control; CXCL = C-X-C motif chemokine; FP = fluticasone propionate; LPS = lipopolysaccharide; poly(I:C) = polyinosinic-polycytidylic acid.Grahic Jump Location
BAL Fluid

BALF was collected at 24 h after the last poly(I:C) treatment as previously reported.15 The BALF was centrifuged at 500 × g for 10 min at 4°C. The cell pellet was resuspended in 0.2% NaCl to induce hemolysis of erythrocytes. After isotonization by adding the same volume of 1.6% NaCl, the total number of BAL cells was counted and aliquoted for flow cytometry analysis. The lung was also removed and stored at −80°C for immunoblotting.

Flow Cytometry Analysis

A cell suspension was incubated with FITC-conjugated anti-macrophage (MOMA2) antibody (2 μg/mL) or FITC-conjugated anti-7/4 antibody (2 μg/mL) and counterstained with propidium iodide (2 μg/mL) to allow exclusion of necrotic cells. The cells were subsequently analyzed using a flow cytometer (ALTRA II; Beckman Coulter Japan).

Immunoblotting and Enzyme-Linked Immunosorbent Assay

The lung tissue was homogenized in a lysis buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.5% sodium deoxycholate, 1 mM DTT, 0.5% Nonidet P-40, and a tablet of protease inhibitors [Roche Diagnostics GmbH]). The sample containing 40 μg protein was separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane. The membrane was incubated with primary antibody (1:500 or 1:3,000) in 5% skim milk TBS/T (20 mM Tris [pH 7.6], 150 mM NaCl, 0.1% Tween 20) at 4°C overnight followed by incubation with secondary antibody for 2 h at room temperature. The immunoreactive bands were visualized by ECL Plus on x-ray film. KC, CXCL10 (IFN-γ-induced protein 10 [IP-10]), IFN-γ, and IFN-β in BALF supernatant was determined using a Quantikine mouse CXCL1/KC, CXCL10/IP-10, and IFN-γ enzyme-linked immunosorbent assay kit (R&D Systems, Inc) and VeriKine mouse IFN-β enzyme-linked immunosorbent assay kit (Pestka Biomedical Laboratories, Inc).

Measurement of Lung Function

At 24 h after the last poly(I:C) dosing, lung function before and 1 min after 5, 10, or 20 mg/mL inhaled histamine or 1, 2.5, or 5 mg/mL inhaled methacholine was measured in the conscious mouse by a two-chambered, double-flow plethysmography system (Pulmos-1; MIPS Ltd.). Airway resistance (sRaw/Vt) was shown as the ratio of the specific airway resistance (sRaw) and the individual tidal volume (Vt) in place of the thoracic gas volume. The airway responsiveness was determined as percent increase of airway resistance (Δ[sRaw/Vt]: %) by comparing before and 1 min after histamine or methacholine inhalation.

Cigarette Smoke Exposure

A/J mice were exposed to cigarette smoke (4% cigarette smoke diluent with compressed air) for 30 min/d for 11 days using the commercially marketed filtered Hi-lite cigarettes (17 mg of tar and 1.4 mg of nicotine per cigarette; Japan Tobacco Inc) and using a Tobacco Smoke Inhalation Experiment System for small animals (Model SIS-CS200; Sibata Scientific Technology Ltd) as described previously.15 Poly(I:C) was administered intranasally bid under anesthesia with 3% isoflurane for 3 days from 24 h after the last cigarette smoke exposure.

Statistical Analyses

Results are expressed as means ± SEM. Multiple comparison was performed by analysis of variance followed by the Dunnett multiple comparison test performed using the PRISM 6 software program (GraphPad Software Inc). The comparison between two groups was performed by unpaired t test with Welch correction or Mann-Whitney test. Statistical significance was defined as P < .05.

Poly(I:C) Induced Airway Inflammation in Mice

In preliminary studies, poly(I:C) (0.1-10 mg/mL intranasally) dose-dependently evoked airway neutrophilia in A/J mice (e-Fig 1, e-Appendix 1). Therefore, we used a submaximal dose of poly(I:C) (1 mg/mL intranasally) in this study.

Intranasal treatment (bid) of poly(I:C) for 1 day significantly increased neutrophils in BALF by 230% compared with vehicle control subjects (neutrophils/mL, 1.8 ± 0.1 × 104 cells/mL to 4.1 ± 0.2 × 104 with poly[I:C]) (Fig 1A). Continuous treatment with poly(I:C) for 2 or 3 days showed further induction by 390% and 570%, respectively. Similarly, CXCL1 production was significantly increased by poly(I:C) on day 1 with a maximal increase on day 2, and this was sustained on day 3 (Fig 1B). Necrotic cells in BALF, mainly dead bronchial epithelial cells, were significantly increased (P < .001) by poly(I:C) (Fig 1C). CXCL10 (IP-10) and IFN-β productions were also significantly increased by poly(I:C) on days 1 to 3, and IFN-γ production was significantly increased by poly(I:C) on days 2 and 3 (e-Fig 2, e-Appendix 1).

Effect of FP and Dexamethasone on Poly(I:C)-Induced Airway Inflammation

As shown in Figure 1A, intranasal FP (0.005-0.5 mg/mL) did not inhibit the poly(I:C)-induced neutrophilia at any time. Similarly, CXCL1 production and the number of necrotic cells in BALF were not influenced by FP treatment (Figs 1B, 1C). The CXCL10, IFN-β, and IFN-γ productions were not reduced by FP either (e-Fig 2, e-Appendix 1). In contrast, FP (0.05 and 0.5 mg/mL) almost completely inhibited lipopolysaccharide (LPS)-induced airway neutrophilia in mice (Fig 1D). In addition, we confirmed that oral dexamethasone (10 mg/kg) did not affect the poly(I:C)-induced airway neutrophilia (e-Fig 3, e-Appendix 1).

Poly(I:C)-Induced AHR

In naive A/J mice, histamine inhalation (20 mg/mL) increased airway resistance determined by Δ(sRaw/Vt) by only 7.3% ± 0.5% (n = 4) compared with that before histamine inhalation. However, the airway response to histamine was significantly increased to 42.2% ± 2.5% (n = 4) after treatment of poly(I:C) on three subsequent days (Fig 2A). This AHR to histamine (5-20 mg/mL) induced by poly(I:C) was not inhibited by FP (Fig 2A). As with histamine, the airway response to methacholine (1-5 mg/mL) was significantly greater after poly(I:C) treatment (Fig 2C). The AHR to methacholine induced by poly(I:C) was not affected by FP (Fig 2C). However, FP (0.05 mg/mL) almost completely inhibited LPS-induced AHR to histamine or methacholine in mice (Figs 2B, 2D).

Figure Jump LinkFigure 2. A-D, Effects of FP on AHR to histamine/methacholine induced by poly(I:C). FP (0.05 mg/mL) was administered intranasally 2 h before intranasal poly(I:C) (1 mg/mL) dosing. AHR was determined as the increment of airway resistance (Δ[sRaw/TV]) before and 1 min after histamine (A, B) or methacholine (C, D) inhalation. AHR to histamine or methacholine induced by poly(I:C) was dose-dependently increased (A, C). On the contrary, AHR to histamine or methacholine induced by LPS (0.1 mg/mL) was completely reduced by FP (0.05 mg/mL) (B, D). Each value is presented as mean ± SEM (n = 3-4). Significant difference from control: ##P < .01 or ###P < .001. Significant difference from poly(I:C) or LPS: ***P < .001. AHR = airway hyperresponsiveness; His = histamine; MCh = methacholine; sRaw = specific airway resistance; TV = tidal volume. See Figure 1 legend for expansion of other abbreviations.Grahic Jump Location

Poly(I:C) also induced AHR to histamine or methacholine in BALB/c and C57BL/6J mice. We also observed that the AHR to histamine or methacholine by poly(I:C) was not affected by FP (e-Fig 4, e-Appendix 1).

TLR3 Expression in Lung Tissue From Cigarette Smoke-Exposed Mice and Effects of Cigarette Smoke Exposure on Poly(I:C)-Induced Inflammation

Western blotting analysis indicated that TLR3 protein expression in the lung tissue from cigarette smoke-exposed mice was significantly greater than that from air-exposed mice (Fig 3A) (TLR3/β-actin ratio: air, 0.18 ± 0.02; smoke, 0.47 ± 0.09; n = 7, P < .01). Furthermore, poly(I:C)-smoke combination increased neutrophils further when compared with cigarette smoke alone (Fig 3B). CXCL1 levels were significantly higher with a combination of poly(I:C) and cigarette smoke by about 40-fold compared with no-smoke no-poly(I:C) control (Fig 3C) and also higher than smoke alone or poly(I:C) alone (Figs 1B, 3C). Similar results were found in necrotic cell numbers (Fig 3D). The neutrophilia, CXCL1 production, and necrotic cell number in combined poly(I:C) with smoke were not inhibited by FP (Figs 3B‐D). In addition, cigarette smoke worsened poly(I:C)-induced AHR to histamine (Fig 4A) and methacholine (Fig 4B).

Figure Jump LinkFigure 3. Expression of TLR3 in the lung tissue and effects of poly(I:C) on airway inflammation induced by cigarette smoke in mice. Mice were exposed to cigarette smoke (4%) for 30 min/d for 11 d. Vehicle, poly(I:C) (1.0 mg/mL), and FP (0.05 mg/mL) were administered intranasally bid for 3 d following the last smoke exposure. Next day, mice were anesthetized, and BAL fluid collection was performed. A, TLR3 and β-actin were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis/Western blotting in the lung tissue from air or cigarette smoke-exposed mice. Upper panel shows a typical immunoblot of TLR3 and β-actin in the lung of air- or cigarette smoke-exposed mice. Lower panel shows the ratio of TLR3 and β-actin, calculated by measuring band density. B, Effect of poly(I:C) on cigarette smoke-exposed animals and effect of FP on BAL neutrophils. C, Effect of poly(I:C) on cigarette smoke-exposed animals and effect of FP on BAL CXCL1 concentrations. D, Effect of poly(I:C) on cigarette smoke-exposed animals and effect of FP on BAL necrotic cells. Each value is presented as mean ± SEM (n = 4-6). Significant difference from air: ##P < .01 (A, B, C) or ###P < .001 (D), and from smoke control: *P < .05 or ***P < .001. TLR = Toll-like receptor. See Figure 1 legend for expansion of other abbreviations.Grahic Jump Location
Figure Jump LinkFigure 4. Effects of poly(I:C) on AHR induced by cigarette smoke. Mice were exposed to cigarette smoke (4%) for 30 min/d for 11 d. Vehicle or poly(I:C) (1.0 mg/mL) were administered intranasally bid for 3 d following the last cigarette smoke exposure. Next day, measurement of lung function was performed. A, AHR was determined as the increment of airway resistance (Δ[sRaw/TV]) before and 1 min after histamine inhalation. B, AHR was determined as the increment of airway resistance (Δ[sRaw/TV]) before and 1 min after methacholine inhalation. Each value is presented as mean ± SEM (n = 3-5). Significant difference from air: ###P < .001, and from smoke control: *P < .05 or ***P < .001. See Figure 1 and 2 legends for expansion of abbreviations.Grahic Jump Location

TLR3 recognizes not only virus-derived dsRNA but also its synthetic analog, poly(I:C), so that poly(I:C) has been used to mimic the response to RNA virus infection.13,14,1621 The in vitro activation of TLR3 by poly(I:C) is reported to induce type 1 IFN secretion via IFN regulatory factor 3,13 which is an innate immune response to protect the host cell.14 In fact, we observed type 1 IFN, IFN-β, and CXCL10 production in BALF by poly(I:C) (e-Fig 2). However, at same time, RNA virus and poly(I:C) are reported to induce the secretion of proinflammatory cytokines and chemokines18,2123 via activation of nuclear factor-κB in airway epithelial cells.13 Ritter and colleagues19 showed that poly(I:C) stimulated a wide range of cytokines from small airway epithelial cells. Stowell and colleagues20 also reported that poly(I:C)-induced recruitment of neutrophils into BAL and airway inflammation was markedly reduced in TLR3 knock-out mice, suggesting poly(I:C)-induced airway inflammation is TLR3 dependent. In the present study, poly(I:C) induced airway neutrophilia and CXCL1 production in naive mice, and repeated poly(I:C) treatment induced severe airway neutrophilia.

RSV infection in mice is reported to induce airway neutrophilia24 and also induce IFN-β,25 CXCL10, IFN-γ,26 KC (mouse homolog of IL-8),27 and AHR.6 Influenza infection also showed AHR and KC production.7,8 In addition, HRV1b infection showed neutrophilia as well as IFN production.28 Thus, the poly(I:C) model closely mimicked RNA virus infection, although virus is replicated and stimulated continuously. Therefore, we treated poly(I:C) bid for 3 days to get stable inflammatory signal.

It is known that inhaled corticosteroid treatment has less benefit on asthmatic exacerbations triggered by respiratory viruses.2933 Gustafson and colleagues34 demonstrated that oral prednisone therapy failed to show any benefit in experimental rhinovirus infection. In addition, de Kluijver and colleagues35 reported that budesonide failed to inhibit cytokine production in nasal lavage during experimental rhinovirus infection in subjects with and without asthma. Recently, allergen-induced inflammation in the presence of RSV infection was reported to be corticosteroid insensitive in mice.10 In the present study, we have shown for the first time, to our knowledge, that poly(I:C)-induced airway inflammation and AHR in naive mice were corticosteroid refractory (Figs 1, 2), which is similar to the virus challenge model. IFN-β, CXCL10, and IFN-γ as well as CXCL1 were not inhibited by FP either (e-Fig 2). As we were able to show that the same dose of FP (0.05 mg/mL) inhibited LPS-induced airway neutrophilia by 99.6% (Fig 1D), it indicated that the dose of FP in the poly(I:C) model was sufficient. Thus, corticosteroid-insensitive inflammation is the another key example to explain that poly(I:C)-induced inflammation is similar to RNA virus-induced inflammation.

As A/J mice are known to be genetically hyperreactive to bronchospasm, we also examined poly(I:C)-induced AHR in BALB/c and C57BL/6J mice, which are more popular strains for respiratory research. As seen in e-Figure 4, poly(I:C) induced AHR in both mouse strains, and the AHR was not inhibited by FP. Therefore, poly(I:C) caused AHR in all strains used in this article.

As previously reported, respiratory virus infection is a common trigger of exacerbations of asthma and COPD, and these patients are also more susceptible to respiratory virus infections.2 Poly(I:C) enhances allergen-induced airway inflammation in ovalbumin-sensitized mice and rats.16,36 Oxidative stress is also reported to enhance TLR3 response in airway epithelial cells.17 As the cigarette smoke exposure in mice is commonly used as an animal model of COPD,37 we investigated the effect of combining of poly(I:C) and cigarette smoking exposure on airway inflammation in mice. As shown in Figure 3A, cigarette smoke exposure up-regulated TLR3 protein expression in mice lung tissue. The combination of smoke exposure and poly(I:C) treatment enhanced airway neutrophilia compared with each treatment alone (Fig 3B). Poly(I:C) also significantly increased CXCL1 production in BALF in cigarette smoke-exposed mice. The combination of cigarette smoke and influenza virus or HRV induces airway neutrophilia38 or a large amount of CXCL8 from human airway epithelial cells,17 reporting agreement with our observations seen in cigarette smoke-exposed mice. Poly(I:C) also significantly increased the number of necrotic cells in BALF of cigarette smoke-exposed mice. It has been demonstrated that TLR3 recognizes not only virus-derived dsRNA but also dsRNA from necrotic cells, which thus serves as an endogenous TLR3 ligand.39 Released dsRNA from necrotic cells may therefore further stimulate TLR3 signaling. Although TLR3 expression was augmented by cigarette smoke exposure in our study, another report did not find any effect of cigarette smoke exposure on the expression of TLR3.40 A different smoke-exposure system/protocol and a different strain of mice may account for the differences in results.

In conclusion, we demonstrated that RNA virus mimic poly(I:C) induced corticosteroid-refractory airway inflammation and AHR via TLR3 signaling. In addition, cigarette smoke enhanced poly(I:C)-induced airway inflammation possibly via an increase in TLR3 expression. These findings suggest that TLR3 stimulation by RNA virus may be involved in corticosteroid-refractory exacerbations of COPD and that this model will be useful for development of novel therapies for corticosteroid-insensitive exacerbations of COPD and asthma.

Author’s contributions: Dr Kizawa had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Dr Kimura: contributed to the study concept and design; acquisition, analysis and interpretation of data; drafting of the manuscript; and approved the final version of the manuscript.

Mr Ueda: contributed to the acquisition, analysis and interpretation of data, and approved the final version of the manuscript.

Mr Eto: contributed to the acquisition, analysis and interpretation of data, and approved the final version of the manuscript.

Mr Watanabe: contributed to the acquisition, analysis and interpretation of data, and approved the final version of the manuscript.

Dr Masuko: contributed to the analysis and interpretation of data, and approved the final version of the manuscript.

Dr Kusama: contributed to the analysis and interpretation of data, and approved the final version of the manuscript.

Dr Barnes: contributed to the interpretation of data, drafting of the manuscript, and approved the final version of the manuscript.

Dr Ito: contributed to the study concept and design; acquisition, analysis and interpretation of data; drafting of the manuscript; and approved the final version of the manuscript.

Dr Kizawa: contributed to the study concept and design; acquisition, analysis and interpretation of data; drafting of the manuscript; and approved the final version of the manuscript.

Financial/nonfinancial disclosures: The authors have reported to CHEST the following conflicts of interest: Dr Barnes receives research funding from and has been on scientific advisory boards for AstraZeneca, Boehringer Ingelheim GmbH, Chiesi Ltd, Cipla Ltd, GlaxoSmithKline plc, Novartis AG, and UCB, all of which are involved in marketing treatments for COPD patients. Dr Ito is currently an employee of RespiVert Ltd and has honorary contract with Imperial College. Dr Kizawa receives research funding from RespiVert Ltd and from the Ministry of Education, Culture, Sports and Technology (MEXT) of Japan. Messrs Ueda, Eto, and Watanabe, and Drs Kimura, Masuko, and Kusama have reported that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Role of sponsors: The sponsors had no role in the design of the study, the collection and analysis of the data, or in the preparation of the manuscript.

Other contributions: This study was performed at the Department of Physiology and Anatomy, Nihon University School of Pharmacy and Airway Disease Section, National Heart and Lung Institute, Imperial College.

Additional information: The e-Appendix and e-Figures can be found in the “Supplemental Materials” area of the online article.

AHR

airway hyperresponsiveness

BALF

BAL fluid

CXCL

C-X-C motif chemokine

dsRNA

double-stranded RNA

FITC

fluorescein isothiocyanate

FP

fluticasone propionate

HRV

human rhinovirus

IFN

interferon

IP-10

interferon γ-induced protein 10

KC

murine CXCL1

LPS

lipopolysaccharide

poly(I:C)

polyinosinic-polycytidylic acid

RSV

respiratory syncytial virus

sRaw

specific airway resistance

TLR

Toll-like receptor

Vt

tidal volume

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To Y, Ito K, Kizawa Y, et al. Targeting phosphoinositide-3-kinase-delta with theophylline reverses corticosteroid insensitivity in chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2010;182(7):897-904. [CrossRef] [PubMed]
 
Kim TB, Kim SY, Moon KA, et al. Five-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside attenuates poly (I:C)-induced airway inflammation in a murine model of asthma. Clin Exp Allergy. 2007;37(11):1709-1719. [CrossRef] [PubMed]
 
Koarai A, Sugiura H, Yanagisawa S, et al. Oxidative stress enhances toll-like receptor 3 response to double-stranded RNA in airway epithelial cells. Am J Respir Cell Mol Biol. 2010;42(6):651-660. [CrossRef] [PubMed]
 
Matsukura S, Kokubu F, Kurokawa M, et al. Role of RIG-I, MDA-5, and PKR on the expression of inflammatory chemokines induced by synthetic dsRNA in airway epithelial cells. Int Arch Allergy Immunol. 2007;143(suppl 1):80-83. [CrossRef] [PubMed]
 
Ritter M, Mennerich D, Weith A, Seither P. Characterization of Toll-like receptors in primary lung epithelial cells: strong impact of the TLR3 ligand poly(I:C) on the regulation of Toll-like receptors, adaptor proteins and inflammatory response. J Inflamm (Lond). 2005;2:16. [CrossRef] [PubMed]
 
Stowell NC, Seideman J, Raymond HA, et al. Long-term activation of TLR3 by poly(I:C) induces inflammation and impairs lung function in mice. Respir Res. 2009;10:43. [CrossRef] [PubMed]
 
Yamashita K, Imaizumi T, Taima K, et al. Polyinosinic-polycytidylic acid induces the expression of GRO-alpha in BEAS-2B cells. Inflammation. 2005;29(1):17-21. [CrossRef] [PubMed]
 
Meusel TR, Kehoe KE, Imani F. Protein kinase R regulates double-stranded RNA induction of TNF-alpha but not IL-1 beta mRNA in human epithelial cells. J Immunol. 2002;168(12):6429-6435. [PubMed]
 
Tsuji K, Yamamoto S, Ou W, et al. dsRNA enhances eotaxin-3 production through interleukin-4 receptor upregulation in airway epithelial cells. Eur Respir J. 2005;26(5):795-803. [CrossRef] [PubMed]
 
Stark JM, Khan AM, Chiappetta CL, Xue H, Alcorn JL, Colasurdo GN. Immune and functional role of nitric oxide in a mouse model of respiratory syncytial virus infection. J Infect Dis. 2005;191(3):387-395. [CrossRef] [PubMed]
 
Jewell NA, Vaghefi N, Mertz SE, et al. Differential type I interferon induction by respiratory syncytial virus and influenza a virus in vivo. J Virol. 2007;81(18):9790-9800. [CrossRef] [PubMed]
 
van Schaik SM, Enhorning G, Vargas I, Welliver RC. Respiratory syncytial virus affects pulmonary function in BALB/c mice. J Infect Dis. 1998;177(2):269-276. [CrossRef] [PubMed]
 
Jafri HS, Chavez-Bueno S, Mejias A, et al. Respiratory syncytial virus induces pneumonia, cytokine response, airway obstruction, and chronic inflammatory infiltrates associated with long-term airway hyperresponsiveness in mice. J Infect Dis. 2004;189(10):1856-1865. [CrossRef] [PubMed]
 
Newcomb DC, Sajjan US, Nagarkar DR, et al. Human rhinovirus 1B exposure induces phosphatidylinositol 3-kinase-dependent airway inflammation in mice. Am J Respir Crit Care Med. 2008;177(10):1111-1121. [CrossRef] [PubMed]
 
Doull IJ, Lampe FC, Smith S, Schreiber J, Freezer NJ, Holgate ST. Effect of inhaled corticosteroids on episodes of wheezing associated with viral infection in school age children: randomised double blind placebo controlled trial. BMJ. 1997;315(7112):858-862. [CrossRef] [PubMed]
 
Ducharme FM, Lemire C, Noya FJ, et al. Preemptive use of high-dose fluticasone for virus-induced wheezing in young children. N Engl J Med. 2009;360(4):339-353. [CrossRef] [PubMed]
 
FitzGerald JM, Becker A, Sears MR, Mink S, Chung K, Lee J; Canadian Asthma Exacerbation Study Group. Doubling the dose of budesonide versus maintenance treatment in asthma exacerbations. Thorax. 2004;59(7):550-556. [CrossRef] [PubMed]
 
Harrison TW, Oborne J, Newton S, Tattersfield AE. Doubling the dose of inhaled corticosteroid to prevent asthma exacerbations: randomised controlled trial. Lancet. 2004;363(9405):271-275. [CrossRef] [PubMed]
 
Panickar J, Lakhanpaul M, Lambert PC, et al. Oral prednisolone for preschool children with acute virus-induced wheezing. N Engl J Med. 2009;360(4):329-338. [CrossRef] [PubMed]
 
Gustafson LM, Proud D, Hendley JO, Hayden FG, Gwaltney JM Jr. Oral prednisone therapy in experimental rhinovirus infections. J Allergy Clin Immunol. 1996;97(4):1009-1014. [CrossRef] [PubMed]
 
de Kluijver J, Grünberg K, Pons D, et al. Interleukin-1beta and interleukin-1ra levels in nasal lavages during experimental rhinovirus infection in asthmatic and non-asthmatic subjects. Clin Exp Allergy. 2003;33(10):1415-1418. [CrossRef] [PubMed]
 
Takayama S, Tamaoka M, Takayama K, et al. Synthetic double-stranded RNA enhances airway inflammation and remodelling in a rat model of asthma. Immunology. 2011;134(2):140-150. [CrossRef] [PubMed]
 
Stevenson CS, Birrell MA. Moving towards a new generation of animal models for asthma and COPD with improved clinical relevance. Pharmacol Ther. 2011;130(2):93-105. [CrossRef] [PubMed]
 
Botelho FM, Bauer CM, Finch D, et al. IL-1α/IL-1R1 expression in chronic obstructive pulmonary disease and mechanistic relevance to smoke-induced neutrophilia in mice. PLoS ONE. 2011;6(12):e28457. [CrossRef] [PubMed]
 
Freudenburg W, Moran JM, Lents NH, Baldassare JJ, Buller RM, Corbett JA. Phosphatidylinositol 3-kinase regulates macrophage responses to double-stranded RNA and encephalomyocarditis virus. J Innate Immun. 2010;2(1):77-86. [CrossRef] [PubMed]
 
Hudy MH, Traves SL, Wiehler S, Proud D. Cigarette smoke modulates rhinovirus-induced airway epithelial cell chemokine production. Eur Respir J. 2010;35(6):1256-1263. [CrossRef] [PubMed]
 

Figures

Figure Jump LinkFigure 1. Effect of a corticosteroid on poly(I:C) induced airway neutrophilia and CXCL1 production in A/J mice. Poly(I:C) (1.0 mg/mL) was administered intranasally bid for 1 to 3 days. A, Effect of FP (0.005, 0.05, and 0.5 mg/mL) on BAL neutrophil numbers. B, Effect of FP (0.005, 0.05, and 0.5 mg/mL) on BAL CXCL1 concentrations. C, Effect of FP (0.005, 0.05, and 0.5 mg/mL) on BAL necrotic cells. Each value is presented as mean ± SEM (n = 4-7). Significant difference from time-matched control: #P < .05 or ##P < .01. FP (0.005, 0.05, and 0.5 mg/mL) was treated at 24 h before the first poly(I:C) administration in addition to at 2 h before every poly(I:C) dosing showing effects on BAL neutrophils (A), and CXCL1 (B). Each value is presented as mean ± SEM (n = 5-6). D, LPS (0.1 mg/mL) was administered intranasally 2 h after FP dosing (0.005, 0.05 and 0.5 mg/mL) and BAL fluid collected 3 h after LPS dosing. Each value is presented as mean ± SEM (n = 5). Significant difference from control: #P < .05, ##P < .01, or ###P < .001, and from LPS: ***P < .001. Con = control; CXCL = C-X-C motif chemokine; FP = fluticasone propionate; LPS = lipopolysaccharide; poly(I:C) = polyinosinic-polycytidylic acid.Grahic Jump Location
Figure Jump LinkFigure 2. A-D, Effects of FP on AHR to histamine/methacholine induced by poly(I:C). FP (0.05 mg/mL) was administered intranasally 2 h before intranasal poly(I:C) (1 mg/mL) dosing. AHR was determined as the increment of airway resistance (Δ[sRaw/TV]) before and 1 min after histamine (A, B) or methacholine (C, D) inhalation. AHR to histamine or methacholine induced by poly(I:C) was dose-dependently increased (A, C). On the contrary, AHR to histamine or methacholine induced by LPS (0.1 mg/mL) was completely reduced by FP (0.05 mg/mL) (B, D). Each value is presented as mean ± SEM (n = 3-4). Significant difference from control: ##P < .01 or ###P < .001. Significant difference from poly(I:C) or LPS: ***P < .001. AHR = airway hyperresponsiveness; His = histamine; MCh = methacholine; sRaw = specific airway resistance; TV = tidal volume. See Figure 1 legend for expansion of other abbreviations.Grahic Jump Location
Figure Jump LinkFigure 3. Expression of TLR3 in the lung tissue and effects of poly(I:C) on airway inflammation induced by cigarette smoke in mice. Mice were exposed to cigarette smoke (4%) for 30 min/d for 11 d. Vehicle, poly(I:C) (1.0 mg/mL), and FP (0.05 mg/mL) were administered intranasally bid for 3 d following the last smoke exposure. Next day, mice were anesthetized, and BAL fluid collection was performed. A, TLR3 and β-actin were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis/Western blotting in the lung tissue from air or cigarette smoke-exposed mice. Upper panel shows a typical immunoblot of TLR3 and β-actin in the lung of air- or cigarette smoke-exposed mice. Lower panel shows the ratio of TLR3 and β-actin, calculated by measuring band density. B, Effect of poly(I:C) on cigarette smoke-exposed animals and effect of FP on BAL neutrophils. C, Effect of poly(I:C) on cigarette smoke-exposed animals and effect of FP on BAL CXCL1 concentrations. D, Effect of poly(I:C) on cigarette smoke-exposed animals and effect of FP on BAL necrotic cells. Each value is presented as mean ± SEM (n = 4-6). Significant difference from air: ##P < .01 (A, B, C) or ###P < .001 (D), and from smoke control: *P < .05 or ***P < .001. TLR = Toll-like receptor. See Figure 1 legend for expansion of other abbreviations.Grahic Jump Location
Figure Jump LinkFigure 4. Effects of poly(I:C) on AHR induced by cigarette smoke. Mice were exposed to cigarette smoke (4%) for 30 min/d for 11 d. Vehicle or poly(I:C) (1.0 mg/mL) were administered intranasally bid for 3 d following the last cigarette smoke exposure. Next day, measurement of lung function was performed. A, AHR was determined as the increment of airway resistance (Δ[sRaw/TV]) before and 1 min after histamine inhalation. B, AHR was determined as the increment of airway resistance (Δ[sRaw/TV]) before and 1 min after methacholine inhalation. Each value is presented as mean ± SEM (n = 3-5). Significant difference from air: ###P < .001, and from smoke control: *P < .05 or ***P < .001. See Figure 1 and 2 legends for expansion of abbreviations.Grahic Jump Location

Tables

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Matsukura S, Kokubu F, Kurokawa M, et al. Synthetic double-stranded RNA induces multiple genes related to inflammation through Toll-like receptor 3 depending on NF-kappaB and/or IRF-3 in airway epithelial cells. Clin Exp Allergy. 2006;36(8):1049-1062. [CrossRef] [PubMed]
 
Yamamoto M, Sato S, Hemmi H, et al. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science. 2003;301(5633):640-643. [CrossRef] [PubMed]
 
To Y, Ito K, Kizawa Y, et al. Targeting phosphoinositide-3-kinase-delta with theophylline reverses corticosteroid insensitivity in chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2010;182(7):897-904. [CrossRef] [PubMed]
 
Kim TB, Kim SY, Moon KA, et al. Five-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside attenuates poly (I:C)-induced airway inflammation in a murine model of asthma. Clin Exp Allergy. 2007;37(11):1709-1719. [CrossRef] [PubMed]
 
Koarai A, Sugiura H, Yanagisawa S, et al. Oxidative stress enhances toll-like receptor 3 response to double-stranded RNA in airway epithelial cells. Am J Respir Cell Mol Biol. 2010;42(6):651-660. [CrossRef] [PubMed]
 
Matsukura S, Kokubu F, Kurokawa M, et al. Role of RIG-I, MDA-5, and PKR on the expression of inflammatory chemokines induced by synthetic dsRNA in airway epithelial cells. Int Arch Allergy Immunol. 2007;143(suppl 1):80-83. [CrossRef] [PubMed]
 
Ritter M, Mennerich D, Weith A, Seither P. Characterization of Toll-like receptors in primary lung epithelial cells: strong impact of the TLR3 ligand poly(I:C) on the regulation of Toll-like receptors, adaptor proteins and inflammatory response. J Inflamm (Lond). 2005;2:16. [CrossRef] [PubMed]
 
Stowell NC, Seideman J, Raymond HA, et al. Long-term activation of TLR3 by poly(I:C) induces inflammation and impairs lung function in mice. Respir Res. 2009;10:43. [CrossRef] [PubMed]
 
Yamashita K, Imaizumi T, Taima K, et al. Polyinosinic-polycytidylic acid induces the expression of GRO-alpha in BEAS-2B cells. Inflammation. 2005;29(1):17-21. [CrossRef] [PubMed]
 
Meusel TR, Kehoe KE, Imani F. Protein kinase R regulates double-stranded RNA induction of TNF-alpha but not IL-1 beta mRNA in human epithelial cells. J Immunol. 2002;168(12):6429-6435. [PubMed]
 
Tsuji K, Yamamoto S, Ou W, et al. dsRNA enhances eotaxin-3 production through interleukin-4 receptor upregulation in airway epithelial cells. Eur Respir J. 2005;26(5):795-803. [CrossRef] [PubMed]
 
Stark JM, Khan AM, Chiappetta CL, Xue H, Alcorn JL, Colasurdo GN. Immune and functional role of nitric oxide in a mouse model of respiratory syncytial virus infection. J Infect Dis. 2005;191(3):387-395. [CrossRef] [PubMed]
 
Jewell NA, Vaghefi N, Mertz SE, et al. Differential type I interferon induction by respiratory syncytial virus and influenza a virus in vivo. J Virol. 2007;81(18):9790-9800. [CrossRef] [PubMed]
 
van Schaik SM, Enhorning G, Vargas I, Welliver RC. Respiratory syncytial virus affects pulmonary function in BALB/c mice. J Infect Dis. 1998;177(2):269-276. [CrossRef] [PubMed]
 
Jafri HS, Chavez-Bueno S, Mejias A, et al. Respiratory syncytial virus induces pneumonia, cytokine response, airway obstruction, and chronic inflammatory infiltrates associated with long-term airway hyperresponsiveness in mice. J Infect Dis. 2004;189(10):1856-1865. [CrossRef] [PubMed]
 
Newcomb DC, Sajjan US, Nagarkar DR, et al. Human rhinovirus 1B exposure induces phosphatidylinositol 3-kinase-dependent airway inflammation in mice. Am J Respir Crit Care Med. 2008;177(10):1111-1121. [CrossRef] [PubMed]
 
Doull IJ, Lampe FC, Smith S, Schreiber J, Freezer NJ, Holgate ST. Effect of inhaled corticosteroids on episodes of wheezing associated with viral infection in school age children: randomised double blind placebo controlled trial. BMJ. 1997;315(7112):858-862. [CrossRef] [PubMed]
 
Ducharme FM, Lemire C, Noya FJ, et al. Preemptive use of high-dose fluticasone for virus-induced wheezing in young children. N Engl J Med. 2009;360(4):339-353. [CrossRef] [PubMed]
 
FitzGerald JM, Becker A, Sears MR, Mink S, Chung K, Lee J; Canadian Asthma Exacerbation Study Group. Doubling the dose of budesonide versus maintenance treatment in asthma exacerbations. Thorax. 2004;59(7):550-556. [CrossRef] [PubMed]
 
Harrison TW, Oborne J, Newton S, Tattersfield AE. Doubling the dose of inhaled corticosteroid to prevent asthma exacerbations: randomised controlled trial. Lancet. 2004;363(9405):271-275. [CrossRef] [PubMed]
 
Panickar J, Lakhanpaul M, Lambert PC, et al. Oral prednisolone for preschool children with acute virus-induced wheezing. N Engl J Med. 2009;360(4):329-338. [CrossRef] [PubMed]
 
Gustafson LM, Proud D, Hendley JO, Hayden FG, Gwaltney JM Jr. Oral prednisone therapy in experimental rhinovirus infections. J Allergy Clin Immunol. 1996;97(4):1009-1014. [CrossRef] [PubMed]
 
de Kluijver J, Grünberg K, Pons D, et al. Interleukin-1beta and interleukin-1ra levels in nasal lavages during experimental rhinovirus infection in asthmatic and non-asthmatic subjects. Clin Exp Allergy. 2003;33(10):1415-1418. [CrossRef] [PubMed]
 
Takayama S, Tamaoka M, Takayama K, et al. Synthetic double-stranded RNA enhances airway inflammation and remodelling in a rat model of asthma. Immunology. 2011;134(2):140-150. [CrossRef] [PubMed]
 
Stevenson CS, Birrell MA. Moving towards a new generation of animal models for asthma and COPD with improved clinical relevance. Pharmacol Ther. 2011;130(2):93-105. [CrossRef] [PubMed]
 
Botelho FM, Bauer CM, Finch D, et al. IL-1α/IL-1R1 expression in chronic obstructive pulmonary disease and mechanistic relevance to smoke-induced neutrophilia in mice. PLoS ONE. 2011;6(12):e28457. [CrossRef] [PubMed]
 
Freudenburg W, Moran JM, Lents NH, Baldassare JJ, Buller RM, Corbett JA. Phosphatidylinositol 3-kinase regulates macrophage responses to double-stranded RNA and encephalomyocarditis virus. J Innate Immun. 2010;2(1):77-86. [CrossRef] [PubMed]
 
Hudy MH, Traves SL, Wiehler S, Proud D. Cigarette smoke modulates rhinovirus-induced airway epithelial cell chemokine production. Eur Respir J. 2010;35(6):1256-1263. [CrossRef] [PubMed]
 
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