Rhinovirus-Induced Exacerbations of Asthma - ATS Journals

Rhinovirus-Induced Exacerbations of Asthma How Is the b2-Adrenoceptor Implicated? Thomas Trian1,2, Lyn M. Moir1,2, Qi Ge1,2, Janette K. Burgess1,2, Curtis Kuo1, Nicholas J. C. King3, Helen K. Reddel2, Judith L. Black1,2, Brian G. Oliver1,2*, and Brent E. McParland1* 1 Respiratory Research Group, Discipline of Pharmacology, and 3Discipline of Pathology, School of Medical Sciences and Bosch Institute, University of Sydney, Australia; and 2Woolcock Institute of Medical Research, Sydney, Australia

Rhinovirus (RV) infections are the major cause of asthma exacerbations in children and adults. Under normal circumstances, asthmatic airway obstruction improves spontaneously or characteristically briskly in response to inhaled b2-adrenergic receptor (b2AR) agonists. During virus-associated exacerbations, an impaired response to b2AR agonists is observed; the reason for this is not known. The objective of this study was to determine the effect of RV infection on airway smooth muscle b2AR function. The human cell line Beas-2B and primary human bronchial epithelial cells (HBECs) were infected with RV (multiplicity of infection 5 1). After 1 or 5 days for primary and Beas-2B cells, respectively, cell culture supernatants were harvested, UV-irradiated to inactivate RV, and applied to human airway smooth muscle cells for 3 days to assess modifications of b2AR function. RV conditioned medium from Beas-2B and HBECs decreased b2AR agonist–induced cAMP by 50 and 65%, respectively (n 5 5; P , 0.05). When cAMP was induced independently of the b2AR using forskolin, no impairment was found. Using flow cytometry, we demonstrated that this decrease was likely the result of b2AR desensitization because membrane but not total cell receptor b2AR was decreased. Pretreatment of HBECs and Beas-2B cells but not human airway smooth muscle cells with the corticosteroids dexamethasone or fluticasone abolished virus-mediated b2AR loss of function. This study shows that epithelial infection with RV induces a decrease of b2AR function on airway smooth muscle cells, potentially explaining the clinical observation of loss of b2AR agonist function during RV-induced asthma exacerbations. Keywords: asthma; rhinovirus; b2-adrenergic receptor; human airway smooth muscle cell

Acute exacerbations of asthma are the major cause of morbidity, mortality, and health care costs related to this disease. Viral respiratory tract infections are the major precipitants of asthma exacerbations in children and adults, but the mechanisms by which they occur are poorly understood (1–3). Of the different virus types associated with exacerbations, rhinoviruses (RV) account for approximately two thirds of the viruses identified (1). Use of regular asthma medications such as corticosteroids 1/2 b2-adrenoceptor agonists (b2-agonists) reduces the overall frequency of asthma exacerbations (4), including those induced by virus (5–7). However, these medications do not prevent virus infections (3), nor do they completely prevent asthma exacerbations from occurring (4).

(Received in original form April 13, 2009 and in final form August 21, 2009) * Joint senior author. This work was supported by NH&MRC Australia and Asthma Foundation of New South Wales. Correspondence and requests for reprints should be addressed to Thomas Trian, Ph.D., Discipline of Pharmacology, The University of Sydney, Sydney, NSW 2006, Australia. E-mail: [email protected] This article contains an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Cell Mol Biol Vol 43. pp 227–233, 2010 Originally Published in Press as DOI: 10.1165/rcmb.2009-0126OC on September 25, 2009 Internet address: www.atsjournals.org

Under normal circumstances, asthmatic airway obstruction improves spontaneously or characteristically briskly in response to inhaled b2-agonists. We first reported objective evidence of loss of circadian recovery of lung function and loss of bronchodilator response to exogenous b2-agonists during exacerbations associated with clinical respiratory infections in a group of patients with asthma whose symptoms and lung function were previously well controlled with corticosteroids (8), These features could be consistent with loss of function of the b2-adrenergic receptor (b2AR); however, the precise etiology of these exacerbations was not known. The lung function changes that were observed during these exacerbations were different from those seen in the same patients before inhaled corticosteroid treatment (8). This indicates that the mechanism by which virusinduced exacerbations occur is different from that resulting from poor asthma control, and we propose that it may be the result of altered b2AR function on airway smooth muscle. In vivo, the epithelial cell is the principal cell type infected by RV in the lower airways (9), and although there is evidence that the underlying submucosal cells are also infected (10), there is no in vivo evidence for direct infection of airway smooth muscle cells. Given that RV-induced inflammation is thought to mediate exacerbations (11), it is highly likely that the epithelial infection is critical in mediating virus-induced exacerbations. In this study, we investigated the hypothesis that RVinduced, epithelial-derived factors down-regulate smooth muscle cell b2AR function. To explore this, we used a coculture system experiment to investigate the effect of factors released from RV-infected epithelial cells upon airway smooth muscle b2AR function.

MATERIALS AND METHODS We established primary cell cultures of human bronchial epithelial cells (HBECs) and human airway smooth muscle cells (HASMCs) from explants of epithelium and airway smooth muscle bundles obtained from macroscopically normal surgical tailings after resection surgery for thoracic lesions or lung transplantation, as previously described (12, 13) (Table 1). HASMCs were used at 80% confluence between passages five and eight. HBECs were used at 100% confluence between passages three and five. HASMCs were maintained in DMEM supplemented with 10% FBS, and HBECs were maintained in bronchial epithelial growth medium (Clonetics, Basel, Switzerland). We also used the human epithelial cell line BEAS-2B (from ATCC, Manassas, VA), which was maintained in 10% FBS in DMEM and used when confluent. Human rhinovirus (RV-16) was grown in Hela cells as previously described (14, 15) and was purified (16) before use in some experiments. RV-conditioned medium was established by the following process. Briefly, RV or UV-inactivated (UVi) RV (multiplicity of infection of 1) was added to the confluent cell monolayers for 1 hour with orbital shaking. The cells were then washed, and the appropriate cell culture medium was added. Conditioned medium was collected 5 days after infection for BEAS-2B cells and 1 day after infection for HBECs. Conditioned medium in the absence of infection (control medium) was also collected. All conditioned media (control, UV, and RV) were UV irradiated before being applied to HASMCs. UV irradiation was performed by placing 250 ml of conditioned medium or RV stock solution

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 43 2010

TABLE 1. PATIENT CHARACTERISTICS

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Smoking Status

Packs per year

Stopped (yr)

Age (yr)

Sex

Medication

FEV1%

FVC1%

Diagnosis

Ex-smoker Current Ex-smoker Ex-smoker Unknown Current Unknown Unknown Ex-smoker Unknown Ex-smoker Unknown Unknown Unknown Unknown Unknown

30 Unknown 40 56 Unknown Unknown Unknown Unknown 60 Unknown 30 Unknown Unknown Unknown Unknown Unknown

23 Unknown 28 , 1 yr Unknown Unknown Unknown Unknown Unknown Unknown , 1 yr Unknown Unknown Unknown Unknown Unknown

78 56 80 77 41 53 53 56 71 80 77 50 36 84 58 56

Female Male Male Male Male Male Male Male Male Female Female Female Female Female Male Male

Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown

1.25 Unknown 72 64 Unknown Unknown Unknown Unknown 2.05 Unknown 1.1 Unknown Unknown Unknown Unknown Unknown

1.65 Unknown 74 89 Unknown Unknown Unknown Unknown 2.6 Unknown 1.7 Unknown Unknown Unknown Unknown Unknown

Small cell carcinoma Adenocarcinoma Squamous cell carcinoma Non–small cell carcinoma Emphysema Emphysema Bronchiectasis Non–small cell carcinoma Squamous cell carcinoma Non–small cell carcinoma Adenocarcinoma Emphysema Primary pulmonary hypertension Carcinoma Idiopathic pulmonary fibrosis Idiopathic pulmonary fibrosis

into each well of a 12-well plate at a distance of 5 cm for 20 minutes from an UV lamp (germicidal lamp G30T8; Sankyo Denki, Kanagawa, Japan). Efficiency of the UV inactivation was determined by RV titration assay (see Figure E5 in the online supplement). All conditioned media were used undiluted. HASMCs were incubated for 3 days with control or RV-conditioned medium, and cytotoxicity and viability were assessed using a MultiToxFluor Multiplex Cytotoxicity Assay (Promega, Madison, WI) according to the manufacturer’s instructions. Evidence of epithelial RV infection was assessed by the measurement of induced IL-6 and IL-8 release as previously described. ELISA kits for IL-6 and IL-8 were purchased from R&D Systems Europe (Abingdon, UK), and experiments were performed according to the manufacturer’s instructions. The detection limit of these assays was 15.6 pg/ml. b2AR function was assessed by the addition of the b-agonist isoprenaline (10 nM to 1 mM and 100 nM), which was chosen because it caused a submaximal response to HASMCs, and the measurements of intracellular cAMP concentration were performed using the a screen cAMP kit (Perkin Elmer, Melbourne, Australia). In some experiments, cAMP production was also measured after 30 minutes of stimulation with forskolin (10 mM). Efficiency of the kit was determined by assessing the down-regulation of the b2ADBR induced by a prolonged treatment with isoprenaline (Figure E2). The level of b2AR expression was assessed using flow cytometry as previously described (17). Briefly, HASMCs (80–90% confluent) were manually scraped from 6-well plates, suspended in Ca21 and Mg21– free PBS/FBS, fixed using a fixation kit (R&D Systems, Minneapolis, MN), blocked for 1 hour with 4% BSA in PBS, incubated for 1 hour on ice with the primary antibody (rabbit anti-human b2–adrenergic receptor; Abcam, Cambridge, MA), washed twice in PBS, and incubated with the secondary antibody (Alexa Fluor 488–conjugated goat anti rabbit antibody; Molecular Probes, Carlsbad, CA). An appropriate isotype control was purchased from Beckman Dickinson (Franklin Lakes, NJ). Cells were then washed again and resuspended in PBS before FACS analysis. Analysis was conducted on a FACSCalibur Sort (Becton Dickinson) using an argon ion laser with an excitation line at 488 nm and CellQuest software. Events were gated on forward and side scatter parameters to include only live cells. There were no differences in scatter between treated and mock-treated samples, and 10,000 events were collected in each live gate. These events were then analyzed for single-color fluorescence (AlexaFluor 488; Invitrogen, Carlsbad, CA) as a measure of receptor expression. Photomultiplier settings were adjusted on the FACSCalibur to enable the isotype labeling fluorescence to appear within the first decade of the fluorescence (X) axis and the specific receptor labeling to appear accordingly within the first and second decades. Data are shown as histograms, each containing 10,000 events (see Figures 2A and 2B). Changes in fluorescence (i.e., receptor expression) were determined by measuring the percentage of events in labeled mock-treated and treated samples above a 95% arbitrary cutoff on the isotype antibody control profile. The effect of corticosteroids on isoprenaline-induced cAMP production in HASMCs was investigated by treating BEAS-2B cells,

HBECs, or HASMCs with dexamethasone (100 nM) (18) or fluticasone (100 nM) (18). In all cell types, corticosteroids were added 1 hour before the addition of virus or conditioned medium; the corticosteroid remained in the epithelial supernatant throughout the infection period. To mimic the effect of bacterial infection on epithelial cells, a range of toll-like receptor (TLR) agonists was applied to BEAS-2B cells and HBECs for 1 hour. The following agonists were used: lipoteichoic acid (5 mg/ml) or lipopolysaccharide (0.5 mg/ml). After 1 hour, the cell medium was changed to remove TLR-agonists from the medium. Medium was removed after 3 days for BEAS-2B cells and 24 hours for HBECs and used as conditioned medium for HASMCs. We used ANOVA with Fisher’s protected LSD analysis to assess the data for all experiments. We judged a P , 0.05 to be significant.

RESULTS Conditioned medium did not induce cytotoxicity in HASMCs (Figure E1). RV infection was assessed by measuring IL-6 and IL-8 release from BEAS-2B cells after 1, 3, and 5 days. RV infection induced an increase in IL-6 and IL-8 release at 1, 3, and 5 days for IL-6 and at 3 and 5 days for IL-8. Because the amount of IL-6 and IL-8 was higher at 5 days after infection, this time point was chosen for subsequent experiments. Because b2AR activation principally mediates relaxation through the generation of cytosolic cAMP, we measured cellular cAMP concentration in HASMCs after stimulation with a bagonist. In HASMCs incubated with conditioned medium from RV-infected BEAS-2B cells, the response to b2AR activation was dramatically attenuated in comparison to the ‘‘no virus’’ control and the UVi control (P , 0.05; n 5 4) (Figure 1A). To confirm that the response observed with BEAS-2B–conditioned medium was comparable to that of primary cells, we infected primary bronchial epithelial cells (HBECs). Similarly, as for BEAS-2B–generated conditioned medium, RV-conditioned medium from HBECs reduced the increase in cAMP due to b2AR activation of HASMCs, whereas UVi-conditioned medium had no effect (Figure 1B). To ensure that the response was specific to RV and not a reaction to other factors in the inoculum, purified RV was also used. A similar and significant decrease of cAMP in response to isoprenaline after RV-conditioned and purified RVconditioned medium was observed (Figure E6). To demonstrate that this impairment was at the level of the b2AR and not at some other step in the pathway, forskolin, a direct activator of cAMP, was used in a similar series of experiments. No impairment in forskolin-induced cAMP concentration was observed in HASMCs treated with HBECs or

Trian, Moir, Ge, et al.: Rhinovirus-Induced Exacerbation of Asthma

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Figure 1. Isoprenaline (100 nM)-induced cAMP response in human airway smooth muscle cells (HASMCs) using conditioned medium from (A) BEAS-2B cells (n 5 5) and (B) human bronchial epithelial cells (HBECs) (n 5 5) and forskolin (10 mM)-induced cAMP response using conditioned medium from (C) BEAS-2B cells (n 5 5) and (D) HBECs (n 5 5). Conditioned medium was made by infecting BEAS-2B cells with rhinovirus for 5 days and HBECs for 24 hours. HASMCs were incubated with conditioned medium for 3 days. C 5 conditioned medium with no virus (open columns); RV 5 rhinovirusconditioned medium (solid columns); UVi 5 UV-inactivated conditioned medium (shaded columns). cAMP was measured using an a screen cAMP kit. *Significant difference from no virus control (P , 0.05).

BEAS-2B RV-conditioned medium in comparison to control and UVi conditioned medium (Figures 1C and 1D). To explore whether the decrease in cAMP was due to a reduced b2AR number, we examined cell surface expression of b2AR using flow cytometry (17). We found a decreased percentage of b2AR-positive cells incubated with RV-conditioned medium from BEAS-2B cells and HBECs compared with the ‘‘no virus’’ control conditioned medium (Figures 2A–2C). Because under certain conditions b2AR can internalize (19), we permeabilized HASMCs so that the total b2AR number could be captured by a detecting antibody. The total b2AR number for HASMCs was not different between the different conditions, indicating that the decreased b2AR function induced by the RVconditioned medium was at least partially attributable due to a decrease in b2AR membrane expression and not because of a decrease in total b2AR number (Figure 2D). Corticosteroids are used for the treatment of asthma and are known to up-regulate b2AR expression, so we assessed whether the RV-induced impaired function of HASMC b2AR could be prevented by previous corticosteroid treatment. When BEAS-2B cells and HBECs were treated with dexamethasone (100 nM) or fluticasone (100 nM) for 1 hour before RV infection, conditioned medium from RV-infected epithelial cells no longer produced an attenuated response to the bAR agonist (Figures 3A and 3C). When we treated HASMCs with dexamethasone (100 nM) or fluticasone (100 nM), and not epithelial cells, RV-conditioned medium induced a small but nonsignificant reduction in b2AR function (Figures 3B and 3D).

To explore whether the factor released from the RV-infected epithelial cells was heat labile, we heated the conditioned medium (508C for 20 min) before applying it to HASMCs. Heating abolished the ability of RV-conditioned medium from HBECs and BEAS-2B cells to attenuate the rise in cAMP caused by b2AR activation (Figure E3A). Moreover, we assessed the effect of direct infection of HASMCs with RV and UVi RV on b2AR function. Neither direct application of UVi-RV for up to 3 days nor the addition of live RV for up to 8 hours attenuated the rise in cAMP caused by b2AR activation (Figure E3B). To investigate if this viral-induced response could involve endogenous prostanoids, we pretreated HASMCs with indomethacin (10 mM) for 1 hour before adding conditioned medium. Indomethacin treatment of HASMCs did not affect the impairment in b2AR-activated cAMP caused by RV infection (Figure E4). To investigate if this viral-induced response could be generalized to other pathogens, such as bacteria, we generated conditioned medium from BEAS-2B cells and HBECs treated with the bacterial toxins lipoteichoic acid or lipopolysaccharide. Neither treatment altered b2AR function in HASMCs (Figures 4A and 4B).

DISCUSSION Our findings for the first time shed some light on why b2-agonists may be less effective during asthma exacerbations caused by rhinovirus infection. We exposed HASMCs to a conditioned

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Figure 2. Effect of BEAS-2B– and HBECconditioned medium on HASMC b2AR expression as assessed by flow cytometry. A and B are representative flow cytometry data for membrane b2AR expression on HASMCs incubated with BEAS-2B– and HBECconditioned medium, respectively. HASMCs are stained with isotype control (black) or b2AR antibody (red and green). HASMCs were incubated with conditioned medium for 3 days. Conditioned medium with no virus (red line) and with RV-conditioned medium (green line). C (n 5 4) and D (n 5 4) represent mean data for the percentage cells with positive b2AR membrane expression and total expression on HASMCs. *Significant difference from no virus control (P , 0.05).

medium taken from epithelial cells infected with RV and tested the response of HASMCs to a b-agonist. When compared with using ‘‘no virus’’ or UVi virus, the medium from RV-infected epithelial cells caused a reduced response to b-agonist, as indicated by a reduced level of intracellular cAMP. This effect was not due to the RV medium. We also found that the mediator released from the epithelial cells was heat labile. Viral infection often results in a secondary bacterial infection, yet the effect of the virus on b2AR function could not be replicated using bacterial toxins, suggesting that the reduction in b2AR function is virus specific. Furthermore, although we used poly I:C only as a surrogate for other viruses, our results suggest that the observed response is rhinovirus specific. The decrease in cAMP was abolished by pretreating the epithelial cells with corticosteroids before the addition of RV but not by pretreating HASMCs. Results obtained from the present study suggest that only infectious RV induces the release of mediators that are capable of reducing the response to a b-agonist in HASMCs. Billington and colleagues found that direct infection of HASMCs also reduced the cAMP response to a b-agonist (isoprenaline) (20). Although the conditioned medium in the present study was UV irradiated before incubation with the epithelial cells, it could be argued that the effect seen was the result of residual RV in the epithelium medium. Because this was a possibility, we directly infected HASMCs with RV for 2, 8, and 24 hours (Figure E3B) but found that at 2 and 8 hours the b2AR-induced cAMP response was not different from the control ‘‘no virus’’ treated cells. At 24 hours, like Billington and colleagues (20), we found a decreased cAMP response, but under our experimental conditions this was likely to be attributable to a large increase in HASMC death. Billington and colleagues also found that even though the b2AR-induced

cAMP response to isoprenaline was decreased as a result of RV infection in HASMCs, the direct activation of adenylyl cyclase by forskolin resulted in an increase in cAMP generation (i.e., adenylyl cyclase sensitization) (20). In our study, forskolininduced cAMP generation was not different between conditions with or without the addition of RV. It is likely, therefore, that in our study the decrease in the b2AR-induced cAMP response was not caused by direct infection of HASMCs. It was most likely due to the effect of some mediators released into the epithelial conditioned medium, which in turn caused the reduced b2AR function in HASMCs. The mediators that caused the decreased b2AR-induced cAMP response remain to be identified, but heating the RVconditioned medium prevented the effect. This result suggests that mediators released by HBECs after an RV infection are at least heat labile. One possible candidate is IL-1b, which has been shown to alter b2AR function (21). In our experiment this is unlikely because there was no detectable increase in IL-1b release in our RV-conditioned medium compared with control conditioned medium (data not shown). Furthermore, although we used poly I:C only as a surrogate for other viruses, our results suggest that the observed response is rhinovirus specific. Poly I:C is often used as a surrogate for virus infection because it activates the viral receptors TLR-3, RIG-I, and MDA5 (22); however, the cellular response to RV infection and replication is likely to be much more complex than this. Activation of the cellular receptor for RV (ICAM-1 and LDL receptor for the major and minor group rhinoviruses, respectively) can induce cellular responses. In the current study, this is unlikely because exposure to UVi rhinovirus did not result in impaired b2-induced cAMP. The cellular response to the detection of double-stranded RNA is also


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Figure 3. Isoprenaline (100 nM)-induced cAMP response in HASMCs using conditioned medium from (A and B) BEAS-2B (n 5 8) and (C and D) HBECs (n 5 8). The effect of corticosteroid treatment (dexamethasone [Dex], 100 nM; fluticasone [Flut], 100 nM) was investigated by treating the epithelial cells for 1 hour before RV infection (A and C) or by treating the HASMCs 1 hour before application of the conditioned medium (B and D). Conditioned medium was made by infecting BEAS-2B cells with RV for 5 days and HBECs with RV for 24 hours. HASMCs were incubated with conditioned medium for 3 days. C 5 ‘‘no virus’’ conditioned medium; RV 5 rhinovirus infection conditioned medium; UVi 5 conditioned medium generated after exposure to UV-inactivated rhinovirus. cAMP was measured using an a screen cAMP kit. *Significant difference from control (P , 0.05).

unlikely to account for the effect observed because treatment with Poly I:C did not affect b2-induced cAMP. It is therefore reasonable to assume that cellular response to the detection of single-stranded RNA through receptors such as TLR7 or viral proteins (e.g., 3c protease) (23) results in the induction of factors that decrease b2-induced cAMP in airway smooth muscle cells. Corticosteroid treatment of the epithelial cells before RV infection abolished the down-regulation of the b2AR in HASMCs. In contrast, corticosteroid treatment of HASMCs did not prevent the effect of medium from the RV-infected epithelium. Our findings are consistent with a case-control study in which the observations were thought to be due to RV infection (24), which showed that children were less likely to experience asthma exacerbations if they had been prescribed inhaled corticosteroids (5). Among adults hospitalized for asthma, RVpositive patients were less likely to be using maintenance inhaled corticosteroids than those who were RV negative (7). Systemic corticosteroids are the mainstay of treatment for severe asthma exacerbations regardless of the cause (25), but there is little evidence for the effectiveness of prednisone or fluticasone in chil-

dren with acute virus-induced wheezing at the onset of common cold symptoms (26, 27). Together, our data and the above studies suggest that patients with asthma should be treated with prophylactic corticosteroid during seasons when the prevalence of common cold is increased to prevent an asthma exacerbation or loss of b2-agonist efficacy. b2AR desensitization is an important mechanism of b2AR regulation. In asthma, a shortacting b2-agonist reduced b2AR membrane expression via a mechanism upstream of protein kinase A (28). However, in the present study, the desensitization was not due to pretreatment with b2-agonists, which demonstrates that other mechanisms can decrease b2AR function. In a recent paper, Moore and colleagues have shown that respiratory syncytial virus infection of HASMCs can also decrease b2AR function (29). However, this was observed in response to direct infection with respiratory syncytial virus. In our study, direct infection of HASMCs with RV did not affect b2AR function. Although it is known that RV infects the nasal tract, it can also infect the airways (9). Evidence for direct infection of HASMCs by RV in vivo is still controversial.

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asthma exacerbation due to rhinovirus infection. Our results also suggest that pretreatment with corticosteroids may be effective in reducing the loss of b2AR function. Author Disclosure: H.R. has received consultancy fees from Biota and Novartis for less than $1,000 each. She has received advisory board fees from AstraZeneca for $10,001 to $50,000, GlaxoSmithKline and Novartis for $1,001 to $5,000, each along with lecture fees from AstraZeneca for $10,000 to $50,000 and from Getz Pharma and Merck Sharp & Dohme for $1,001 to $5,000 each. She has received institutional grants from GlaxoSmithKline and AstraZeneca for $5,001 to $10,000 each. B.G.O. received an industry-sponsored research grant from Merck Sharp & Dohme for $10,001 to $50,000. J.B. received a sponsored grant from National Health and Medical Research Council Australia and Cooperative Research Centre for Asthma and Airways for more than $100,001 each and has patents pending/received from Cooperative Research Centre for Asthma and Airways. None of the other authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

References

Figure 4. Isoprenaline (100 nM)-induced cAMP response in HASMCs using conditioned medium from (A) BEAS-2B cells (n 5 4) and (B) HBECs (n 5 4). To mimic bacterial infection, the following toll-like receptor agonists were applied to the epithelial cells for 1 hour and then incubated for 5 days in growth medium: lipopolysaccahride (LPS), lipoteichoic acid (LTA), and the synthetic polyinosinic-polycytidylic acid double-stranded RNA (Poly:IC). Conditioned medium was made by treating BEAS-2B cells for 5 days and HBECs for 24 hours. HASMCs were incubated with conditioned medium for 3 days. C 5 control conditioned medium; RV 5 RV-conditioned medium; UVi 5 conditioned medium generated in response to UV-inactivated rhinovirus. cAMP was measured using an a screen cAMP kit. *Significant difference from control (P , 0.05).

Our finding in the present study is very exciting with respect to understanding why b2-agonists may be less efficacious during a RV-induced asthma exacerbation. However, there remain several unanswered questions that need to be addressed in future studies, in particular the nature of the factor(s) in conditioned medium. In conclusion, this study shows that epithelial infection with RV induces decreased b2AR function of airway smooth muscle cells, which potentially explains why RV-induced asthma exacerbations occur. Moreover, our results can account for the clinical observation that b2-agonists can be less effective during an

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