Anknuten till Forskning
PhD-student in Mikael Adner's group at IMM/EAAF
MD, currently intern physician at Karolinska University Hospital
Asthma is a chronic obstructive airway disease characterised by breathlessness, coughing, chest tightness and wheezing, affecting approximately 10% of Swedish population with 5% of them having severe asthma1. The pathophysiologic mechanisms behind the symptoms are bronchoconstriction, airway hyperresponsiveness (AHR), airway inflammation, airway remodelling and mucous hypersecretion. AHR is a process of exaggerated bronchoconstriction to stimuli that normally don’t affect healthy individuals and is a consequence of airway smooth muscle (ASM) contraction. Exercise-induced bronchoconstriction (EIB) can be a feature of asthma but AHR can also occur in healthy athletes. EIB is considered to be caused by increased osmolarity at the epithelial barrier after vigorous breathing causing loss of water. Damage or irritation of the airway epithelium is an important trigger of asthma symptoms, and thus also of EIB2. Epithelial damage can lead to secretion of many different mediators, among them Interleukin (IL)-33, thymic stromal lymphopoietin (TSLP) and IL-25, collectively named epithelial alarmins, which cause downstream activation of adaptive and innate immune system as well as of activation of local structural cells3.
Mast cells (MCs) are essential effector immune cells in asthma being great contributors to the pathophysiology of the disease. MCs are activated by many different stimuli to migrate to the lung, where they can be triggered by inflammatory stimuli, antigens or mechanical stress to degranulate and secrete multiple pro-inflammatory mediators, both pre-formed and de-novo synthesised4. MCs are intertwined with ASM cells, which are the effectors of bronchoconstriction and there is substantial evidence that MCs are crucial for EIB5. Mediators released from activated MC are also potent airway constrictors, such as histamine that is stored in MC granules, and prostaglandins and leukotrienes which are lipid mediators that are rapidly produced after activation of the MCs4.
Epithelial alarmins have to a large extent been studied in different animal models in context of asthma. However, findings in animal models have not always been consistent with the relatively few findings in human models. In a clinical trial blocking TSLP markedly reduced not only the bronchoconstriction that followed an allergen challenge, but also the accompanying airway inflammation6. Therefore, a hypothesis emerged that epithelial alarmins released during epithelial damage are the common denominator for both AHR and airway inflammation in asthma. The aim of my thesis is therefore to elucidate the role of epithelial alarmins in asthma, and particularly in EIB, and asthma attacks induced by other stimuli such as allergen, with focus on studies in humans as well as in human cell and tissue models.
Tumour-free surgical lung specimens are obtained with patient consent at Karolinska University Hospital, Solna, from patents undergoing pulmonary surgery, with permission from regional ethical review board in Stockholm (ref. no. 2018/1819-31/1). At the IMM airway pharmacology laboratory in Biomedicum, bronchial segments are dissected out from the specimen under a microscope and the bronchial segments are cut into rings with diameter <2 mm. The rest of the lung tissue can be used to extract lung MCs. The human bronchial (HB) rings as well as lung MCs can be cultured for several days, allowing for controlled exposure to IL-33, TSLP and IL-25. Some pilot experiments have been performed using guinea pig trachea (GPT) with permission from regional ethical review board in Stockholm (ref. no. N143-14, 10973-2019). GPT can be cut to rings and cultured similarly to the human bronchi. Some animals are sensitised to ovalbumin prior to harvesting the trachea in order to mimic an allergic asthma setting. HB rings and rings from GPT are mounted on to a myograph in an organ bath and changes in isometric tension as response to contractile mediators are measured. Generated contractile responses of an airway ring are represented as percentage maximal contraction of that specific ring.
Contractile mediators that act directly on ASM, include histamine, leukotrienes and prostaglandins. Mediators that exert indirect effect, on the other hand, must first activate the MC to release mediators, and those mediators subsequently affect the ASM tone. Whether they are attached to the HB ring or cultured, MCs can be activated by stimulation of the IgE-receptor with anti-IgE antibody or in the case of guinea pigs with the antigen ovalbumin, but also by increase in osmolarity using mannitol. In the organ bath, MC activation is studied by analysing the contractile response of the HB ring and by analysing the release of histamine and lipid mediators into the organ bath fluid. In culture, MC activation and degranulation can also be studied by measuring release of mediators as well as by analysing expression of a protein, CD63, on the cell surface, by using fluorescence-activated cell sorting (FACS). Measurement of lipid mediators is performed using liquid chromatography with mass spectrometry (LC-MS/MS) in collaboration with the laboratory of Craig Wheelock at MBB.
Inhalation of mannitol, along with eucapnic voluntary hyperpnea (EVH) are used to establish presence and severity of EIB in human subjects. The Unit for Clinical Asthma and Allergy Research, Karolinska University Hospital Huddinge has an internationally leading setting for studies of mechanisms in asthma using lung function testing and bronchial provocations together with experimental medicine interventions and collection and analysis of body fluids.
The alarmins are the next targets for new biologic drugs to treat asthma, but the mechanisms of their potential anti-asthmatic effects are still unclear7. This project aims to understand the role of epithelial alarmins in asthma, with focus on EIB, a condition restricting many individuals, not only persons with asthma diagnosis, from keeping an active an active lifestyle. The project will in addition to defining alarmin mechanisms also enable exploration of the lipid mediator profile of mannitol-induced MC activation, which has not been done before, which will extend the understanding of mechanisms in EIB. The studies will furthermore shed light on the potential interactions between the individual epithelial alarmins, which have been indicated by some studies, but disproven by others.
1. Backman H, Jansson SA, Stridsman C, et al. Severe asthma-A population study perspective. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology 2019;49:819-28.
2. Hallstrand TS, Moody MW, Aitken ML, Henderson WR, Jr. Airway immunopathology of asthma with exercise-induced bronchoconstriction. The Journal of allergy and clinical immunology 2005;116:586-93.
3. Roan F, Obata-Ninomiya K, Ziegler SF. Epithelial cell-derived cytokines: more than just signaling the alarm. The Journal of clinical investigation 2019;129:1441-51.
4. Bradding P, Arthur G. Mast cells in asthma--state of the art. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology 2016;46:194-263.
5. Dahlén B, Roquet A, Inman MD, et al. Influence of zafirlukast and loratadine on exercise-induced bronchoconstriction. The Journal of allergy and clinical immunology 2002;109:789-93.
6. Gauvreau GM, O'Byrne PM, Boulet LP, et al. Effects of an anti-TSLP antibody on allergen-induced asthmatic responses. The New England journal of medicine 2014;370:2102-10.
7. Papi A, Brightling C, Pedersen SE, Reddel HK. Asthma. Lancet (London, England) 2018;391:783-800.