The Influence of Asthma Control on the Severityof Virus-Induced Asthma Exacerbations

2 The influence of asthma control on the severity of virus-induced 3 asthma exacerbations

4 David J. Jackson MRCP MSc PhD,a,b,c Maria-Belen Trujillo-Torralbo BSc,a,b,c Jerico

5 del-Rosario BSc,a,b,c Nathan Bartlett PhD, a,b Michael R. Edwards PhD,a,b Patrick

6 Mallia MRCP PhD, a,b,c Ross P. Walton PhD,a,b and Sebastian L. Johnston FRCP

7 PhD.a,b,c

8 aAirway Disease Infection Section, National Heart and Lung Institute, Imperial

9 College, London, UK; bMRC & Asthma UK Centre in Allergic Mechanisms of Asthma;

10 cImperial College Healthcare NHS Trust.

12 Corresponding author: Dr David J Jackson, Airway Disease Infection Section,

13 National Heart and Lung Institute, Imperial College London, Norfolk Place, London

14 W2 1PG, United Kingdom. [email protected]. Tel: +442075943764, fax:

15 +442072628913.

17 Funding: This study has been funded by the European Research Council (ERC FP7

18 grant 233015), MRC Centre grant G1000758 and NIHR BRC Centre grant P26095.

19 20 21 Capsule summary 22 Rhinovirus is the dominant trigger for asthma exacerbations. This data demonstrates 23 that the level of asthma control at the time of a rhinovirus infection directly influences 24 the severity and duration of a virus-induced exacerbation. 25 26 Key words 27 Rhinovirus; asthma control; exacerbation 28 29 Abbreviations 30 RV: Rhinovirus 31 ACQ: Asthma control questionnaire

32 FEV1: Forced expiratory volume in 1 second 33 PCR: Polymerase chain reaction 34 VL: Virus load 35 36 37 To the Editor, 38 39 Our understanding of asthma control has evolved in recent years to include both the 40 idea of current control (gauged mostly from symptoms), as well as a link with future 41 risk, notably exacerbations.1 Despite currently available therapies, many patients 42 remain uncontrolled and asthma exacerbations, which are most frequently triggered 43 by rhinovirus infections, remain a major unmet need.2 The development of human 44 experimental models of rhinovirus-induced asthma exacerbations have led to 45 important advances in our understanding of exacerbation pathogenesis, 3 however to 46 date studies have been limited through inclusion of well-controlled asthmatics only.3 47 It is therefore unknown how the level of control at the time of infection influences the 48 severity of the exacerbation, however studies of naturally-occurring exacerbations 49 support a relationship between control and exacerbation frequency.4,5 50 We therefore analysed the relationship between the degree of baseline control in 51 asthmatics infected with rhinovirus-16 (RV16) and clinical measures of exacerbation 52 severity following inoculation. A comparison of clinical outcomes between asthmatic 53 and non-asthmatic subjects including the same subjects as reported herein has been 54 published elsewhere.6 55 Twenty-eight adult asthmatic volunteers were recruited (Table 1). The study was 56 approved by a local research ethics committee (09/H0712/59) and all volunteers 57 provided written informed consent to participate. The degree of asthma control at 58 baseline was assessed using the Juniper Asthma Control Questionnaire (ACQ-7) 59 with 3 control groups defined by ACQ cut-offs of ≤0.75 for well-controlled (n=12), 60 0.76-1.49 for partially-controlled (n=8) and ≥1.5 for uncontrolled asthma (n=8) 61 respectively.7 Full inclusion/exclusion criteria are available in the online supplement. 62 Subjects were seen at baseline and on days 2,3,4,5,7,10 and 42 post-inoculation

63 with RV16. Daily diary cards of upper and lower respiratory symptoms and FEV1 64 measurements were commenced 2 weeks prior to baseline sampling and continued 65 for 6 weeks.6 The lower respiratory score was calculated from scores graded 0–3 for 66 cough on waking; wheeze on waking; daytime cough; daytime wheeze; daytime 67 chest tightness; daytime shortness of breath; nocturnal cough, wheeze or shortness 68 of breath.6 Bronchial mucosal lining fluid was sampled for measurement of a range 69 of cytokines and chemokines including IL-4, IL-5, IL-6, IL-13, IL-33, CXCL10/IP-10 70 and CXCL11/ITAC using the technique of bronchosorption.6 This involves passing a 71 specially designed probe with a synthetic absorptive tip down the operating port of a 72 bronchoscope. Further details regarding study design, methodology and statistical 73 analyses are provided online and have been previously published.6

74 Following RV16 infection, we observed an increase in upper and lower respiratory 75 symptoms across all asthma control categories. However the uncontrolled 76 asthmatics (ACQ ≥1.5) experienced significantly greater virus-induced respiratory 77 symptoms than those with superior baseline control. Specifically, although upper 78 respiratory symptoms reached a similar magnitude across the groups, the 79 uncontrolled asthmatics experienced a more prolonged cold than the other 80 asthmatics (Fig. 1A).

81 Analysis of lower respiratory symptom scores also revealed substantial differences 82 between control categories: Uncontrolled asthmatics experienced a greater maximal 83 level of lower respiratory symptoms on day 4 (P<0.05) and a slower recovery with 84 significant differences in symptoms observed on days 8-11 and 13 between control 85 groups (all P<0.05) (Fig. 1B). Analysis of the total lower respiratory symptom score 86 (summation of daily scores during the 14 days post-inoculation - a more complete 87 measure of exacerbation severity), demonstrated that uncontrolled asthmatics had a 88 significantly greater mean score (59.8±8.6) than asthmatics with better baseline 89 control: ACQ≤0.75, total score 21.3±5.5 (P=0.001); ACQ 0.76-1.49, total score 90 32.5±6.3 (P=0.02)(Fig. 1C). Importantly, the virus-induced increases in lower 91 respiratory symptom scores were corrected for pre-infection levels and were 92 therefore those over and above any baseline symptoms for each subject (see online 93 methods).

94 The relationship between baseline control and virus-induced symptom severity was

95 mirrored by changes in lung function: Analysis of daily FEV 1 measurements 96 highlighted that uncontrolled asthmatics had significantly greater virus-induced falls

97 in FEV1 from baseline: 24.6±3.1% compared to 16.3±1.7% for subjects with an ACQ 98 of 0.76-1.49 (P=0.025), and 14.9±2.0% for the well-controlled group (P=0.021) 99 (Fig.1D). Taken together, the uncontrolled asthmatics experienced a more severe 100 and prolonged exacerbation than asthmatics with better baseline control.

101 Categorising the asthmatics by asthma severity (defined by GINA8) rather than 102 asthma control revealed significantly greater virus-induced lower respiratory 103 symptoms in the moderately-severe asthmatics (n=17) compared to the mild 104 asthmatics (n=11) (P=0.01, Fig. 1E). However, analysis of the moderately-severe 105 asthmatics alone demonstrated that the observed relationship between control and 106 exacerbation severity persisted within this single severity category (r=0.5, P=0.04) 107 (Fig. 1F). A significant relationship was also evident when the correlation was limited 108 to asthmatics on inhaled corticosteroid (ICS) therapy (r=0.58, P=0.02). Interestingly, 109 no relationship in asthma was seen between lower respiratory symptoms and

110 baseline FEV1 (r=-0.14, P=0.49). Unfortunately due to the small number of subjects 111 in each control category it has not been possible to address the potential for 112 confounding factors further and future larger studies are required to investigate this. 113 In addition it should be noted that the majority of the well-controlled asthmatics (8/12) 114 had mild asthma.

115 Measurement of a range of Th1 (CXCL10/IP-10 and CXCL11/ITAC), Th2 (IL-4, IL-5, 116 IL-13, IL-33) and pro-inflammatory (IL-6) cytokines/chemokines in bronchial mucosal 117 lining fluid was performed at baseline and on day 4 post-inoculation and analysed 118 according to asthma control group (Supplementary Table 1). Overall, there were no 119 significant differences across control groups for these mediators; however, we were 120 interested to note that the most marked virus-induced increases in the Th1/anti-viral 121 chemokines CXCL10/IP-10 and CXCL11/ITAC were seen in the well-controlled 122 asthmatics (Supplementary Table 1). In addition, virus load (VL), was measured in 123 nasal lavage samples at 6 time-points between day 2 and 10 post-inoculation as well 124 as in BAL on day 4. At each of these time-points median VL was numerically greater 125 in the uncontrolled asthmatics compared to either of the other groups; however these 126 differences did not reach significance (supplement Table 2).

127 To our knowledge this study is the first to analyse the influence of asthma control on 128 the outcome of a rhinovirus infection in asthma. We observed more severe 129 exacerbations in uncontrolled asthmatics irrespective of asthma severity or treatment 130 status. These results support findings by Bateman demonstrating the influence of 131 control on the risk of future exacerbations,5 as well as findings of the GOAL study in 132 which unscheduled healthcare utilisation for exacerbations related to the level of 133 control achieved rather than the treatment received.4

134 We acknowledge that the small sample size (8-12 per group) makes it difficult to 135 draw significant mechanistic conclusions from our study. However, the higher levels 136 of Th2 cytokines and viral load and the finding of less marked Th1/anti-viral induction 137 in asthmatic subjects compared to healthy controls observed reproducibly in our 138 earlier reports3,6,9 cannot explain why uncontrolled asthma is associated with greater 139 rhinovirus infection-induced asthma symptoms, as trends in these responses did not 140 reach statistical significance in the numbers studied (Supplementary Tables 1 &2). 141 We plan to address this critical gap in knowledge in future studies using high 142 throughput discovery approaches and greater numbers of poorly controlled subjects. 143 Interestingly a recent trial of inhaled interferon-β in asthma10 identified significant 144 improvements in the outcome of naturally-occurring respiratory virus infections in 145 more severe and poorly-controlled asthmatics only.

146 The data presented here extend our existing understanding of the link between the 147 two domains of asthma control – current control and future risk - and further highlight 148 the importance of maintaining adequate control in reducing the likelihood of severe 149 asthma exacerbations.

150 151 Acknowledgements: This study has been funded by the European Research

152 Council (ERC FP7 grant 233015), MRC Centre grant G1000758 and NIHR BRC

153 Centre grant P26095.

154 David J. Jackson MRCP PhD,a,b,c Maria-Belen Trujillo-Torralbo BSc,a,b,c Jerico del-

155 Rosario BSc,a,b,c Nathan Bartlett PhD, a,b Michael R. Edwards PhD,a,b Patrick Mallia

156 MRCP PhD, a,b,c Ross P. Walton PhD,a,b and Sebastian L. Johnston FRCP PhD.a,b,c

157 aAirway Disease Infection Section, National Heart and Lung Institute, Imperial

158 College, London, UK; bMRC & Asthma UK Centre in Allergic Mechanisms of Asthma;

159 cImperial College Healthcare NHS Trust.

160 161 References

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194 195 Table 1 Baseline demographic and clinical characteristics of study volunteers.

Characteristic Well controlled Partially Uncontrolled P value ACQ: ≤0.75 Controlled ACQ ≥1.50 Across all Between groups (N = 12) ACQ: 0.76-1.49 (N = 8) groups (N = 8) Age (yr) 33±12 38±10 36±10 0.542 -

Sex (%) 0.087 - Female 75 50 25 Male 25 50 75 White ethnicity 10 (83) 5 (63) 7 (88) 0.426 - (% of subjects)

Baseline FEV1 well v’s part: 0.001 Percent of predicted value 95±10 78±7 82±10 0.001 well v’s un: 0.02 part v’s un: 1.0 Asthma severity (as defined by GINA) 0.013 well v’s part: 0.31 Mild asthma (% of subjects) 8 (66.7) 3 (37.5) 0 (0) well v’s un: 0.01 Moderate asthma (% of subjects) 4 (33.3) 5 (62.5) 8 (100) part v’s un: 0.23

Baseline histamine PC20 (mg/mL) 1.55±1.95 0.29±0.61 1.78±2.78 0.277 -

Baseline asthma control (ACQ) 0.56±0.27 1.18±0.15 1.86±0.32 <0.001 well v’s part: <0.001 well v’s un:<0.001 part v’s un: <0.001 Use of inhaled corticosteroids 4(33) 4(50) 7 (88) 0.063 - (% of subjects)

Dose of inhaled corticosteroids (Beclometasone/equivalent (mcg) Median 450 350 400 0.71 - Interquartile range 250-875 200-875 200-500 IgE IU/mL Median 95 356 152 0.183 - Interquartile range 65-212 122-1105 52-710 BAL Eosinophilia (%) Median 0.3 1.3 0.3 0.225 - Interquartile range 0-1.0 0.3-3.0 0-1.8

197 198

199 Figure 1. The response to rhinovirus infection in asthma according to baseline 200 control status. Daily upper (A) and lower (B) respiratory symptoms are shown over 201 the 14 days post-inoculation with rhinovirus. The daily lower respiratory scores have 202 been corrected for baseline symptoms and any effect of bronchoscopy. The total 203 lower respiratory symptom score is the summation of the daily scores over 14 days 204 (C) and the maximal fall in FEV1 (as a percentage change from baseline) (D) are 205 shown for all subjects according to ACQ group. The total lower respiratory symptom 206 score is also shown according to asthma severity (E) with the relationship between 207 ACQ and total lower respiratory symptoms shown for moderately-severe asthmatics 208 only (F). Results shown as lines are mean values (A-E). Statistically significant 209 differences shown in A and B refer to differences across all groups (one-way Anova). 210 Clinical data is missing for 1 well-controlled asthmatic (n=11). *,P<0.05; 211 **,P<0.01;***,P<0.001