Rhinovirus Induces Th2 cytokines and chemokines in the Airways in Asthma David Jackson Clinical Research Fellow Department of Respiratory Medicine National Heart and Lung Institute Imperial College London

Introduction Asthma is a chronic condition characterised by reversible airflow obstruction, airway hyper-responsiveness, and airway inflammation Affects 1 in 12 of the UK population NHS costs are ~£1bn/yr Asthma exacerbations are the major cause for morbidity, mortality and healthcare costs in asthma Respiratory viruses are the most frequent trigger for exacerbations Rhinovirus identified in the majority of episodes Johnston BMJ 1995 … many studies since.

Human Model of Rhinovirus Induced Acute Exacerbations of Asthma ↑ RV-induced lower airway involvement in mild asthmatics compared to healthy volunteers ↑symptoms, ↓lung function and ↑AHR. - Corne, Lancet 2002; Message, PNAS 2008 RV

Human Model of Rhinovirus Induced Acute Exacerbations of Asthma Healthy N = 14 Mild Well controlled Steroid naïve N = 14 Moderate Poorly controlled On maintenance inhaled corticosteroids N = 18 Infection confirmed by demonstration of RV16 RNA by RT-PCR in nasal lavage and serum titre of RV-16 specific antibodies ≥ 1:4 on day 42. This is important because this is the group of patients new treatments are developed for. N = 11 N = 11 N = 17

Human Model of Rhinovirus Induced Acute Exacerbations of Asthma (PC)20 (PC)20 BAL Bronchial brushings Bronchial biopsies Bronchosorption RV 16 Day -15 -14 0 2 3 4 5 7 10 42 Daily spirometry and symptom scores throughout study Nasal lavage and Nasosorption at every visit

‘Nasosorption’ and ‘Bronchosorption’: A new technique for measuring nasal and bronchial mucosal lining fluid. Accurate measurement of proteins in nasal lavage and BAL is extremely difficult. Variable recovery of saline Variable dilutions Many proteins below limits of detection BAL can lead to bronchospasm in asthmatics Large proportion of patients complain of a fever following BAL In paediatrics even nasal lavage considered too invasive and difficult for some age groups. I’m going to leave RV for a moment and talk about one of my other study objectives. 15 pg/ml of IL-X becomes 0.15 pg/ml when dilute in 100ml of saline.

Nasosorption Synthetic Absorptive Matrix (SAM) ‘Leukosorb’ ( Pall Life Sciences) 50mcl of epithelial lining fluid 5 ml of saline is a 100 fold dilution Accuwik Ultra medium is a fibrous material constructed of pure hydroxylated polyester. The fiber surfaces have been modified to be inherently and permanently water wettable. Conjugate Release As a conjugate pad, this material possesses consistent flow characteristics with uniform wicking rates. The hydroxylated nature of the membrane ensures homogenous uptake of the conjugate along with rapid release, facilitating lower volumes of conjugate needed. As manufacturers of diagnostic tests continue to increase the sensitivity of the assays produced, they become increasingly concerned with how artifacts from the materials affect the assay. Many conjugate release materials are manufactured with surfactant or binders which affect conjugate release, sample flow rate, and, ultimately, assay performance. The absence of surfactant and the consistent nature of the Accuwik Ultra medium makes it an ideal choice for use in lateral flow assays as a conjugate release pad. At Pall, we understand how material variation and inefficient conjugate release increase your diagnostic assay manufacturing costs. The Accuwik Ultra medium has been designed with numerous features that make it the superior material choice. Yielding near 100% conjugate release, Accuwik Ultra medium may reduce reagent cost and scrap rates during manufacturing. In addition, this membrane has been designed with low protein binding characteristics that will reduce concerns of critical protein biomarker binding as well as increased test signal intensities. Sample Collection and Storage As diagnostic applications are moved from the clinic to remote field locations, the need to collect and transport samples is increasing. The highly absorbent nature of Accuwik Ultra medium results in an excellent tool for direct collection of biological samples. A unique feature of Accuwik Ultra medium is that it enables stability during sample storage. It also allows for rapid and efficient release of clinically relevant protein biomarkers suitable for diagnostic analyses. Following storage and release from the Accuwik Ultra material, the protein profiles are not altered or degraded, which results in a robust material for clinical sample collection, transport, and storage.  

Bronchosorption Device

Bronchosorption RML bronchus RLL bronchus Sheath SAM advanced Absorbing mucosal lining fluid

Allergic asthma considered a Th2 mediated disease Th1 inflammation induced following viral infection in both healthy and asthmatic subjects RV Kraft M. NEJM 2011

Bronchial CCL22 (MDC) correlates with upper and lower respiratory symptoms during RV-infection in asthma

Increased CCL22 (MDC) and CCL17 (TARC) during RV infection with greatest levels in asthma Median 25 H, 50 asthma

RV Kraft M. NEJM 2011

IL-5 is induced by RV in both the upper and lower airway in asthma

Th2 cytokines induced by RV in asthma IL-13 IL- 4

IL-13 correlates with RV-induced symptoms in asthma

No difference in induction of IFN-gamma (Th1) between asthma and healthy subjects

IL-33 RV Initiator of Type 2 inflammation Induction by influenza in mice. Chang, Nat Immunol 2011 RV IL-33 is a novel member of the IL-1 cytokine family. Soluble IL-33 is known to increase the secretion of Th2 cytokines (IL-4, IL-5, and IL-13) (1) and to act as a chemoattractant for Th2 lymphocytes both in vivo and in vitro, elucidating its ability to induce a Th2 immune response in different inflammatory diseases (4).

IL- 33 correlates with Th2 cytokines and chemokines during RV infection in asthma

IL-33 correlates with upper and lower airway symptoms in asthma

Is RV induction of Th2 pathways in asthma clinically relevant? Mepolizumab (anti-IL-5 mAb) → fewer severe exacerbations in subjects with severe refractory eosinophilic asthma. (RR 0.57; p = 0.02) Haldar, Pavord NEJM 2009 Lebrikizumab (anti-IL-13 mAb) → rate of exacerbations 60% lower in ‘high-Th2’ subgroup only (p = 0.03). No significant effect on other asthmatics. Corren, Matthews NEJM 2011 Jonathan Corren IgG4 humanised monoclonal antibody that specifically binds to IL-13 and inhibits its function

Baseline levels of Th2 cytokines predict levels during exacerbation Great potential to use nasosorption in the clinic to identify suitable patients for anti- IL-5 / anti IL-13 drugs

Summary Bronchosorption and nasosorption allows measurement of previously undetectable proteins Induction of IL-33 and the Th2 pathway by virus in asthma in vivo and relationships to clinical outcomes provides explanation for effectiveness of anti-IL-5 and anti-IL-13 mAb’s in preventing exacerbations in selected asthmatics Baseline levels of IL-5 and IL-13 predict magnitude of induction by virus – possibility to identify suitable asthmatics for mAb therapies Correlation between upper and lower airway protein levels at baseline and following RV infection in asthma Asthma is heterogeneous – new treatments needs to be targeted And finally – don’t underestimate how heterogeneous asthma is.

Acknowledgements Prof Sebastian Johnston Dr Trevor Hansel Belen Trujillo-Torralbo Jerico del Rosario Johnston group Dr Onn Min Kon Hunt Developments Ltd Novartis GSK Belen as well as Jerico who I put through hell by making them assist my 7am bronchoscopies 3 times a week for the best part of a year not to mention the 500 screening visits and the subsequent 460 study visits that they usually helped me with.

Correlation between Upper and Lower Airway Protein Levels in Asthma   Bronchial D4 Nasal D2 Nasal D3 Nasal D4 IL-33 + - IL-5 +++ IL-13 ++ IL-17 IFN-g IL-15 I-TAC IP-10 IL-6 IL-8 TNFa TNF R2 MDC TARC Eotaxin Eotaxin-3 RANTES IL-2 IL-10 MCP-1 MCP-4 GM-CSF MIP-1a MIP-1b MIP-3a IL-12p40 IL-1b IL-16 +, p = <0.05 ++, p = <0.01 +++, p = <0.001

Lower Respiratory Symptoms * P <0.05 Mild compared to moderate asthma

Change in PEF * P <0.05, ** P <0.01 Poorly-controlled compared to well-controlled # P <0.05, Poorly-controlled compared to partially-controlled