Phenotyping Circulating and Lung Resident Eosinophils in Severe Asthma (P-CLESA)

Background: Severe asthma has been defined as "asthma which requires treatment with high dose inhaled corticosteroids (ICS) plus a second controller (and/or systemic corticosteroids) to prevent it from becoming 'uncontrolled' or which remains 'uncontrolled' despite this therapy" (1), representing a most difficult group of participants to manage. It is a heterogeneous condition and the use of mathematical approaches led to the definition of clinical subtypes (phenotypes) from cohorts in Europe, USA and Asia. With the introduction of monoclonal antibody therapies such as anti-IgE antibody (omalizumab), anti-Interleukin (IL)-5/anti-IL5Rα antibody (Mepolizumab, reslizumab and benralizumab) and anti-IL4Rα (dupilumab), personalised medicine has been initiated as these therapies are effective in patients with allergic and/or in asthma patients with increased numbers of a particular type inflammatory cell called an eosinophil (eosinophilic asthma), respectively. Introduction of these targeted therapies has helped a proportion of participants with severe asthma particularly those with eosinophilic severe asthma, but further progress will come with a better understanding of all the driving pathways underlying these phenotypes.

The biology and function of the Eosinophil Eosinophils are characterised by the presence of specific secondary granules, in their cytoplasm, containing toxic cationic proteins(2). Research studies on eosinophil biology indicate that eosinophils function in many ways including cytokine production and the development of airway inflammation. Under physiological conditions, only small numbers of eosinophils are released from the bone marrow. However, eosinophil production is dramatically increased as a result of so-called Th2 cell responses associated with allergic diseases such as asthma (2-4). There is also evidence that eosinophils contribute to homeostatic immune processes (4, 5). This increase in eosinophil production is driven by a dedicated set of cytokines, namely IL-3, IL-5, and granulocyte macrophage-CSF (GM-CSF) (2, 4). Amongst these, the Th2-associated cytokine IL-5 is the most specific cytokine for the eosinophil lineage and is responsible for the expansion of eosinophils from committed bone marrow progenitors, their release into the blood and their survival following migration into the tissues(2, 4, 6). At the site of injury, eosinophils can release their cytotoxic granule proteins as well as preformed cytokines and lipid mediators, to exacerbation of inflammation and tissue damage, which is particularly deleterious when Th2 responses are directed against allergens (2, 4).

Recruitment of eosinophils from the circulation requires blood eosinophils to become activated, leading to their attachment to activated endothelium and movement of white blood cells from the capillaries to the tissues surrounding them (extravasation(7, 8)). Arrest of eosinophils in vessels and their extravasation into the airway wall and through the bronchial tissue and epithelium to the airway lumen are mediated by a set of proteins called integrins(7, 8). Blood eosinophils from subjects with allergy or asthma, have a greater degree of adhesion or migration compared to those from normal volunteers (9).

Pharmacodynamic biomarkers for Mepolizumab treatment response Mepolizumablizumab binds with high specificity and affinity to human IL-5(10), the key T2 cytokine responsible for regulation of blood and tissue eosinophils(11). Mepolizumab prevents IL-5 from binding to the IL-5 receptor complex expressed on the eosinophil cell surface and thus inhibits IL-5 signaling, blocking eosinophil survival and proliferation. Although the exact mechanism of action of IL-5 inhibitors is not fully elucidated, the desired goal is to neutralize the effect of activated eosinophils in blood and tissues and lung, particularly the lung for asthma in order to achieve therapeutic effect.

In asthma, an association between eosinophil overexpression and asthma severity was made in 1990(12) and subsequent studies have since repeated the view that blood, or sputum eosinophil counts can be used to characterize participants with severe eosinophilic asthma (13). In the SIRIUS trial Mepolizumab at a dose of 100 mg administered subcutaneously was shown to reduce the requirement for daily oral corticosteroids while at the same time improving asthma control, lung function, and quality of life and reduced exacerbations(14). In the MENSA trial participants treated with Mepolizumab reported significantly reduced exacerbations and an improvement in asthma control questionnaire scores(15). The Mepolizumab investigators identified blood eosinophils, rather than sputum eosinophils, as a good predictor of the clinical efficacy of Mepolizumab, providing an accessible biomarker for severe eosinophilic asthma. Moreover, secondary analysis(16) of the DREAM(17) and MENSA(15) studies identified clinical efficacy of Mepolizumab in severe asthma participants with baseline eosinophil counts of ≥150-300 cells/µl and ≥300 cells/µl.

Subsets of eosinophils in asthma In mice subsets of eosinophils have been identified(18): i) lung resident eosinophils (rEos) that are IL-5-independent cells with a ring-shaped nucleus and ii) inflammatory eosinophils (iEos), which were defined as IL-5-dependent cells with a segmented nucleus. In a murine model of asthma, Mesnil and colleagues reported that the features of lung rEos remained unchanged but rEos were co-localised with iEos in the lung and that mice lacking rEos had an increased Th2 cell response to inhaled allergens. Studies in man reported that parenchymal rEos found in non-asthmatic human lungs were phenotypically distinct from the iEos isolated from the sputa of eosinophilic asthmatic patients. These data highlight the existence of distinctive circulatory and lung resident subsets of eosinophils in man.

Eosinophils secrete many molecules involved in asthma pathology including cytotoxic granule constituents cytokines and chemokines such as, lipid mediators leukotrienes(2, 19). While the role of IL-5 is a confirmed in eosinophil activation and asthma, the comparative activities of other cytokines such as GM-CSF and IL-3, also implicated in asthma, are yet to be fully determined. Eotaxins (CCL11 and CCL24) are considered to be important chemokines involved in the recruitment of eosinophils into the airways while IL-4 and IL-3 are reported to be overexpressed in the airways of severe asthmatics(20, 21). In addition to these Th2-type cytokines, IFNs and TNFα also upregulate eosinophils and prolong their survival(22, 23). Furthermore, the receptors TSLPR, IL33R and CCR3 present on activated eosinophils were found to be elevated in severe eosinophilic asthma (24) Thus, an investigation of cytokine stimulation of subsets of eosinophils, the possible crosstalk between stimulated eosinophils and the resulting differential mediator release and protein expression will likely provide a better understanding of the underlying pathophysiological signaling pathways involved in severe asthma.

Observational Study This will be an observational study of participants diagnosed with severe asthma being followed up and managed according to agreed UK guidelines for management of severe asthma. In this group, participants will have been treated with Mepolizumab according to the blood eosinophil count. These participants will be recruited and phenotyped according to eosinophilic molecular phenotype by analysis of blood and lung transcriptomics and proteomics. Participants will be evaluated at 0, 3 and 12 months. Incidental clinical finding will be forwarded to the clinical team and GP, as warranted.

Study Rationale The relationship between subsets of circulating and lung resident eosinophils in severe asthma and anti-IL-5 therapy efficacy has not been explored.

Objectives To determine the transcriptomic and mediator profiles of eosinophils from the circulation and the airway, specifically blood and tissue resident, in participants with severe asthma at baseline and on Mepolizumablizumab therapy.

Study Model Unbiased study to identify all subsets of circulating and lung resident eosinophils in biologic naïve participants with severe asthma at baseline and at time-points post treatment with Mepolizumab (anti-IL-5 Rx). Severe asthma participants (Eos>300/ul) will be recruited from the Severe Asthma clinic at the Royal Brompton Hospital, and, as much as feasible, participants will not be on OCS therapy.

Primary Objectives (i) Phenotype subsets of circulating eosinophils from participants with severe asthma at one time-point (ii) Phenotype subsets of circulating and lung eosinophils from participants with severe asthma on Mepolizumab therapy over one year.

Study 1: Phenotype subsets of circulating eosinophils in participants with severe asthma at one time-point

Recruit: 15 biologic naïve SA not on maintenance OCS and 15 SA currently on Mepolizumab therapy with good clinical response.

Blood samples (90ml) will be taken from these participants. Blood eosinophils will be isolated from these samples which will then be subtyped (phenotype) by simultaneously measuring gene and cell surface protein expression in the same cell. Blood eosinophils will also be cell-cultured in the laboratory and stimulated with a variety of cytokines and chemokines the release of inflammatory mediators determined. Together, this will increase our understanding of the cellular heterogeneity in asthmatic eosinophils and identify the difference between peripheral blood eosinophil populations in untreated and Mepolizumablizumab treated participants .

Study 2: Phenotype subsets of circulating and lung eosinophils from participants with severe asthma on Mepolizumab therapy over one year.

Recruit 36 severe asthma participants appropriately characterised severe asthmatics (Eos>300/ul), not on OCS maintenance therapy, to achieve paired biopsy samples from 30 participants

Blood samples will be taken at baseline, 3 and 12 months post-Mepolizumab Therapy. Blood eosinophils will then be isolated and phenotyped by simultaneously measuring gene and cell surface protein expression in the same cell. Blood eosinophils will also be cell-cultured in the laboratory and stimulated with a variety of cytokines and chemokines the release of inflammatory mediators determined. Together, this will increase our understanding of cellular heterogeneity in asthmatic eosinophils post Mepolizumab Therapy.

Bronchoscopy will be performed on 36 participants, sampling endobronchial lung biopsy at baseline and 1 yr post Mepolizumab Therapy; the second procedure will be performed within 2 weeks of the last Mepolizumab injection. Single-cell RNA-seq 10xGenomics and bulk -RNA -seq will be performed, using the optimized protocol of the Gronigen group, on lung tissue eosinophils through enzymatic disassociation of lung tissue (at 4oC) at baseline and 1yr post Mepolizumab therapy. Immunohistochemistry (IHC) will also be performed to characterise cellular content and structure. Four biopsies will be reserved for RNA-seq and two for IHC.