Karolinska Institutet , Stockholm , Sweden AIRWAY SMOOTH MUSCLE AS A TARGET IN ASTHMA : NEW INSIGHTS INTO BRONCHORELAXATION AND HYPERREACTIVITY Martijn Manson Stockholm 2015

Patients with asthma suffer from reversible airway obstruction accompanied by airway hyperresponsiveness (AHR). Airway smooth muscle (ASM) is central to bronchoconstriction and AHR and is therefore the target of existing and potential new therapies for asthma. Activation of bitter taste sensing type 2 receptors (TAS2Rs) on ASM was recently suggested to induce bronchorelaxation by a novel mechanism. Therefore, the first aim of this thesis was to determine the potential of TAS2Rs (and their respective agonists) as possible therapeutic targets on ASM. A secondary aim of this thesis was to assess whether inflammation induced by IL-13 can alter ASM function in intact human airways. The methodology used in this thesis mainly involved the study of isometric contractions and relaxations in smooth muscle preparations from guinea-pigs and humans. The measurement of intracellular Ca 2+ and analyses of mRNA expression following interventions (cytokines/siRNA) was performed in cultured ASM cells. Defined TAS2R agonists including chloroquine, denatonium, dextromethorphan, noscapine and quinine were found to strongly relax guinea pig trachea, pre-contracted by muscarinic agonists. Although the TAS2R agonists were less potent bronchorelaxants than β2-adrenoceptor agonists, they were equally (or more) efficacious following stronger pre-contractions. Chloroquine relaxed antigeninduced contractions, whereas denatonium failed to relax other types of pre-contractions. TAS2R agonists were shown to induce relaxation via distinct mechanisms that do not involve conventional pathways. Human pulmonary arteries, expressing TAS2R mRNA, were relaxed by chloroquine, dextromethorphan and noscapine, indicating that TAS2R agonists also affect vascular smooth muscle function. In guinea pig aorta, TAS2R agonists elicited relaxations following noradrenergic precontractions, independently of the endothelium, BK or L-type Ca2+ channels. Again, responses towards TAS2R agonists were shown to be dependent on the agents used for pre-contraction. Studies in guinea pig taenia coli demonstrated antagonism by denatonium and quinine at α-adrenoceptors. In human ASM cells, TAS2R mRNA expression was decreased by TNFα and glucocorticoids, and increased following 24-48 hours of serum-deprivation. Chloroquine, dextromethorphan, noscapine and quercetin induced relaxation of intact human airways, but differentially inhibited histamineinduced intracellular Ca 2+ mobilisation. The siRNA-induced decrease of TAS2R14 mRNA could not prevent quercetin-mediated inhibitions in human ASM cells. In human small airways, IL-13 induced a 2.5-3.1-fold increase in the potency of carbachol and histamine contractions, without affecting their amplitude. In addition, IL-13 impaired relaxant responses towards salbutamol, but not those towards formoterol, chloroquine and noscapine. IL-13 increased both the potency and the amplitude of histamine-induced intracellular Ca 2+ mobilisation in human ASM cells. The IL-13-induced hyperreactivity of human bronchi and ASM cells was not prevented by the glucocorticoid dexamethasone. In summary, although TAS2R agonists exert powerful relaxations in smooth muscle, their effects in ASM are not mediated by common pathways. As a consequence, the TAS2Rs may be questioned as a target for bronchorelaxation in asthma. On the other hand, IL-13 induces functional remodelling of the ASM in human airways that can contribute to AHR. LIST OF SCIENTIFIC PAPERS This thesis is based on the following publications: I. Pulkkinen V, Manson ML, Säfholm J, Adner M, Dahlén S-E. The bitter taste receptor (TAS2R) agonists denatonium and chloroquine display distinct patterns of relaxation of the guinea pig trachea Am J Physiol Lung Cell Mol Physiol. 2012 Dec 1;303(11):L956-66. II. Manson ML, Säfholm J, Al-Ameri M, Bergman P, Orre AC, Swärd K, James A, Sven-Erik Dahlén, Mikael Adner Bitter taste receptor agonists mediate relaxation of human and rodent vascular smooth muscle Eur J Pharmacol 2014, 2014 Oct 5;740:302-11 III. Manson ML * , James A * , van Rheenen M, Jager M, Zuccaro L, Pulkkinen V, Dahlén S-E, Adner M Effects of bitter tastants on human airway smooth muscle in relation to TAS2R expression Manuscript * These authors contributed equally IV. Manson ML, James A, Säfholm J, Al-Ameri M, Bergman P, Orre AC, Dahlén S-E, Adner M IL-13 induces glucocorticoid-insensitive hyperreactivity of human small airways Manuscript submitted During my time at Karolinska Institutet, I contributed to two additional research studies that were not included in this thesis Säfholm J, Manson ML, Bood J, Delin I, Orre AC, Bergman P, Al-Ameri M, Dahlén SE and Adner M Prostaglandin E2 inhibits mast cell-dependent bronchoconstriction in human small airways via the EP2 receptor J Allergy Clin Immunol, accepted for publication Larsson OJ, Manson ML, Starkhammar M, Fuchs B, Adner M, Kumlien Georén S, Cardell LO TLR7 agonists relax guinea pig airways independently of TLR7: a possible role for quinoline moieties Manuscript submitted CONTENT Background . 1 1.1 Asthma . 1 1.2 Pathophysiology . 2 1.2.1 Heterogeneous disease . 2 1.2. 2 Epithelium . 2 1.2. 3 Airway inflammation – Th2 type . 3 1.2. 4 Airway remodelling . 3 1.3 Airway hyperresponsiveness . 4 1.4 Smooth muscle . 4 1.5 Airway smooth muscle . 5 1.6 Airway smooth muscle contraction . 6 1.7 Airway smooth muscle relaxation . 7 1.8 Taste sensing type 2 receptors . 8 1.8. 1 General . 8 1.8. 2 Extra-oral functions . 8 1.8. 3 TAS2R agonists . 9 1.9 IL-13 . 10 2 Materials and Methods . 11 2.1.1 General . 11 2.1.2 Animals . 11 2.1.3 Human lung tissue . 11 2.1.4 Organ culture . 12 2.1.5 Organ bath – in vitro pharmacology . 12 2.1.6 Airway smooth muscle cell culture . 13 2.1.7 RNA/cDNA isolation and synthesis . 13 2.1.8 Real-time polymerase chain reaction (PCR) . 13 2.1.9 Small interfering RNA (siRNA) . 14 2.1.10 Measurements of mobilisation intracellular Ca 2+ . 15 2.1.11 Enzyme-linked immunosorbent assay (ELISA). 16 2.1.12 Calculations and statistics . 16 3 Aims of the Thesis . 17 4 Results and discussion . 19 4.1 Characterisation of effects TAS2R agonists . 19 4.1.1 TAS2R agonist selection . 19 4.1.2 Airway smooth muscle . 19 4.1.3 Vascular smooth muscle . 22 4.1.4 Gastrointestinal smooth muscle . 23 4.1.5 Detrusor smooth muscle . 24 4.1.6 Summary . 26 4.2 Mechanisms underlying smooth muscle relaxation . 27 4.2.1 Cyclic nucleotides (cAMP/cGMP) . 27 4.2. 2 Hyperpolarization via BK channels . 27 4.2. 3 Localised induction of intracellular Ca 2+ . 28 4.2. 4 Inhibition of bronchoconstrictor-induced Ca 2+ induction . 28 4.2.5 Inhibition of IP3 signalling .