Biologic therapies targeting IL-5 or its receptor reduce eosinophilia and exacerbations in asthma. Breakthrough eosinophilic attacks are usually considered biologic failure. We report 2 patients with severe asthma receiving mepolizumab or benralizumab who developed relapsing hypereosinophilia associated with Pseudomonas aeruginosa airway infection. Case 1 was a 71-year-old woman in Canada with severe eosinophilic asthma and COPD overlap whose eosinophils suddenly rebounded to 1,400 cells/μL during an acute exacerbation while on mepolizumab, coinciding with P aeruginosa infection and resolving after antibiotic treatment. Case 2 was a 56-year-old woman in the United Kingdom with late-onset asthma who developed marked eosinophil rebound (1,690 cells/μL) during benralizumab therapy, again associated with P aeruginosa infection and resolving after antipseudomonal therapy. These cases suggest that P aeruginosa may drive eosinophilia through IL-5-independent mechanisms. Sputum cultures should be considered during exacerbations in patients treated with anti-IL-5 or IL-5R therapies, regardless of type 2 biomarkers.

BACKGROUND:Targeted type 2 (T2) biologics have transformed asthma care, but the clinical response to biologic therapy varies between patients. OBJECTIVE:We sought to assess airways inflammation in T2-high asthmatic patients treated with anti-IL-5 biologics to investigate whether differential mechanism of airway inflammation explains varied response to biologics. METHODS:Proteomic analysis (Olink, 1463 protein panel) and high-sensitivity cytokine analysis (ELISAs) were performed on induced sputum from T2-high severe asthmatic patients in the UK multicenter Mepolizumab EXacerbation study. Samples included were pre-mepolizumab (n = 28), stable on mepolizumab (n = 43), and at first exacerbation (n = 26). RESULTS:Clustering of sputum proteins while stable on mepolizumab identified 2 clusters. Cluster 1 had increased differentially expressed sputum proteins pre-mepolizumab, stable on mepolizumab, and at exacerbation. Patients in cluster 1 were younger at diagnosis, had a longer duration of asthma, lower FEV1%, and higher 5-Question Asthma Control Questionnaire score on mepolizumab. Cluster 1 had increased expression of proinflammatory cytokines (IL-1β, IL-6, and soluble IL-6R), epithelial alarmins (thymic stromal lymphopoietin [TSLP] and IL-33), and neutrophil activation (myeloperoxidase [MPO], neutrophil elastase [NE], and neutrophil extracellular trap concentration [NET]). All patients were T2-high with no difference in fractional exhaled nitric oxide, eosinophil number, or activity (eosinophil-derived neurotoxin, EDN) across the 2 clusters. CONCLUSIONS:In a cohort of T2-high severe asthmatic patients, a subgroup of patients with long duration of disease had worse clinical parameters, increased sputum proteins with increased markers of neutrophil activity, proinflammatory cytokines, and epithelial alarmins even when stable on mepolizumab. This suggests the presence of biology not treated by targeted T2 biologics, which may contribute to poorer outcomes on biologics and could be a treatable airways trait in severe asthma.

Background: Mepolizumab is an anti-IL-5 mAb treatment for severe eosinophilic asthma that reduces asthma exacerbations. Residual airway inflammation with mepolizumab therapy may lead to persistent exacerbations. Oral corticosteroids remain the main treatment for these residual exacerbations. Objective: Our study aimed to explore the corticosteroid responsiveness of airway inflammation after mepolizumab treatment to find potentially treatable inflammatory mechanisms beyond the IL-5 pathway. Methods: The MAPLE trial was a multicenter, randomized, double-blind, placebo-controlled, crossover study of 2 weeks of high-dose oral prednisolone treatment at stable state in 27 patients treated with mepolizumab for severe eosinophilic asthma. We analyzed paired sputum (n = 16) and plasma (n = 25) samples from the MAPLE trial using high-throughput Olink proteomics. We analyzed additional sputum proteins using ELISA. Results: In patients receiving mepolizumab, prednisolone significantly downregulated sputum proteins related to type 2 inflammation and chemotaxis including IL-4, IL-5, IL-13, CCL24, CCL26, EDN, CCL17, CCL22, OX40 receptor, FCER2, and the ST2 receptor. Prednisolone also downregulated cell adhesion molecules, prostaglandin synthases, mast cell tryptases, MMP1, MMP12, and neuroimmune mediators. Neutrophilic pathways were upregulated. Type 2 proteins were also downregulated in plasma, combined with IL-12, IFN-g, and IP-10. IL-10 and amphiregulin were upregulated. Conclusions: At stable state, prednisolone has broad antiinflammatory effects on top of mepolizumab. These effects are heterogeneous and may be clinically relevant in residual exacerbations. (J Allergy Clin Immunol 2024;154:1146-58.)

Background Mepolizumab is an anti-interleukin-5 monoclonal antibody for severe eosinophilic asthma (SEA). The additional effects of prednisolone to mepolizumab, on molecular mechanisms in the airways and blood are poorly understood. Aim Determine the transcriptomic and proteomic effects of prednisolone versus placebo on the airways and blood in patients with SEA treated with mepolizumab. Methods MAPLE was a randomized, double-blind, placebo-controlled crossover trial of prednisolone at stable state in adults with SEA after mepolizumab (Yang F, JACI Pract 2022;10:2925–34.e12). Prednisolone had a minor effect on FEV1 but not on symptoms. Sputum and blood samples were taken before and after high dose prednisolone and placebo in patients treated with mepolizumab. These underwent O-link expression analysis of 1536 proteins. A paired comparison of normalised protein expression for 1536 proteins in sputum and serum were compared in a linear mixed effects model, with Benjamini-Hochberg correction for multiple testing. Nasal scrape samples were taken for transcriptomic analysis after prednisolone and placebo in patients treated with mepolizumab. RNA was extracted (Qiagen) and good quality samples sequenced (Illumina Novaseq). We identified differentially expressed genes with paired t-tests with Benjamini-Hochberg correction for multiple testing. Results 21 participants had paired serum, and 14 had paired sputum, before and after both prednisolone and placebo. Prednisolone significantly downregulated 173 and 229 proteins and upregulated 63 and 140 proteins in sputum and serum respectively. Downregulated proteins in sputum included IL-4, IL-5, IL-13, chemokines, and signatures of mast cells, prostaglandin synthesis, and alternatively activated macrophages. Up-regulated proteins included FKBP5, typical of steroid treatment. 6 people had paired nasal epithelial samples comparing prednisolone to placebo. 28 genes were down-regulated by prednisolone included leukocyte chemotaxis, mast cell tryptase and the 15-lipoxygenase pathway. Conclusions Prednisolone in addition to mepolizumab suppresses type-2 pathways unaffected by IL-5 inhibition in the sputum and blood proteome, and nasal transcriptome. These findings support the notion that the type-2 airway epithelium remains active in mepolizumab-treated patients. The relationship of these additional effects to longer term clinical outcome is unknown. Please refer to page A286 for declarations of interest related to this abstract.

Mepolizumab is an anti-interleukin-5 monoclonal antibody treatment for severe eosinophilic asthma (SEA) that reduces asthma exacerbations. Residual airway inflammation on mepolizumab may lead to persistent exacerbations. Oral corticosteroids have broad anti-inflammatory effects and remain the main treatment for these residual exacerbations. Our study aimed to explore the nature and corticosteroid-responsiveness of airway inflammation after mepolizumab treatment to find potentially treatable inflammatory mechanisms. The MAPLE trial was a multi-centre, randomized, double-blind, placebo-controlled, crossover study of 2 weeks of high-dose oral prednisolone treatment at stable state in patients treated with mepolizumab for SEA. We analysed sputum and plasma samples from the MAPLE trial using high-throughput Olink proteomics. We also analysed plasma microRNA, sputum proteins using ELISA, and nasal mucosal bulk RNA sequencing. In patients receiving mepolizumab, prednisolone significantly downregulated sputum proteins related to type-2 inflammation and chemotaxis including IL-4, IL-5, IL-13, CCL24, CCL26, EDN, CCL17, CCL22, OX40 receptor, FCER2, and the ST2 receptor. Prednisolone also downregulated cell adhesion molecules, prostaglandin synthases, mast cell tryptases, MMP1, MMP12, and neuroimmune mediators. Tissue repair and neutrophilic pathways were upregulated. Type-2 proteins were also downregulated in plasma, combined with IL-12, IFN-γ, and IP-10. IL-10 and amphiregulin were upregulated. In the nasal transcriptome, prednisolone suppressed genes involved in leucocyte chemotaxis, mast cell tryptase, 15-lipoxygenase and MMP12. By contrast, mepolizumab differentially regulated only Galectin-10 in plasma and no sputum proteins, and in nasal tissue affected genes related to cilia, keratinisation, extracellular matrix formation, and IL-4/13 signalling. At stable state, prednisolone has broad anti-inflammatory effects on top of mepolizumab.### Competing Interest StatementIH has received a conference travel grant from GSK. FY and SED have received speaker fees from AstraZeneca. JC, VB, and EM report no declarations of interest. AA is currently an employee of AZ. PJM has received support to attend educational meetings from Chiesi. JB has received personal fees from NuvoAir, and a research grant to his Institute from AstraZeneca, outside the submitted work. CB has received speakers fees from AZ and GSK and has received advisory board fees from AZ. LH has received grants from GSK, Astra Zeneca, Roche/Genentech, has given lectures supported by Astra Zeneca, Sanofi, Circassia, GlaxoSmithKline, has received travel grants from AstraZeneca and GSK, and has honoraria for Advisory Board Meetings from Novartis, Roche/Genentech, GSK, Teva and Celltrion. IDP has received speakers honoraria for speaking at sponsored meetings from Astra Zeneca, Boehringer Inglehiem, Aerocrine, Almirall, Novartis, Teva, Chiesi, Sanofi/Regeneron, Menarini and GSK and payments for organising educational events from AZ, GSK, Sanofi/Regeneron and Teva. He has received honoraria for attending advisory panels with Genentech, Sanofi/Regeneron, Astra Zeneca, Boehringer Ingelheim, GSK, Novartis, Teva, Merck, Circassia, Chiesi and Knopp and payments to support FDA approval meetings from GSK. He has received sponsorship to attend international scientific meetings from Boehringer Ingelheim, GSK, Astra Zeneca, Teva and Chiesi. He has received a grant from Chiesi to support a phase 2 clinical trial in Oxford. He is co-patent holder of the rights to the Leicester Cough Questionnaire and has received payments for its use in clinical trials from Merck, Bayer and Insmed. In 2014-5 and 2019-20 he was an expert witness for a patent dispute involving Astra Zeneca and Teva. CEB has received grants and consultancy fees from 4D Pharma, Areteia, AstraZeneca, Chiesi, Genentech, GlaxoSmithKline, Mologic, Novartis, Regeneron Pharmaceuticals, Roche and Sanofi. RC has received lecture fees from GSK, AZ, Teva, Chiesi, Sanofi and Novartis; honoraria for Advisory Board Meetings from GSK, AZ and Celltrion; sponsorship to attend international scientific meetings from Chiesi, Sanofi and GSK and a research grant to her Institute from AZ for a UK multi-centre study. TSCH has received grants from the Wellcome Trust, grants from The Guardians of the Beit Fellowship, and grants from the NIHR Oxford Biomedical Research Centre during the conduct of the study; and grants from Pfizer Inc., grants from University of Oxford, personal fees from Astra Zeneca, personal fees from TEVA, personal fees from Peer Voice outside the submitted work. ### Clinical TrialThis study is registered on Clinicaltrials.gov ([NCT03610685][1]).### Funding StatementThis study was funded jointly by the Medical Research Council (MRC) UK (MR/M016579/1) and industrial partners within the MRC Refractory Asthma Stratification Programme consortium and by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC). Proteomics and transcriptomics analysis was funded by GSK (ID: 215294). TSCH is supported by a Wellcome Trust Fellowship (211050/Z/18/z). All authors had full access to the full data in the study and accept responsibility to submit for publication. ### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:The study was approved by the Medical Ethics Committee (West of Scotland Research Ethics Service 3, Reference number 18/WS/0060). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAnonymised patient level data analysed and presented in this study are available from the corresponding author on reasonable request, providing the request meets local ethical and research governance criteria after publication. Data will be available immediately after publication for 10 years. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT03610685&atom=%2Fmedrxiv%2Fearly%2F2024%2F02%2F15%2F2024.02.14.24302812.atom

Macrolides reduce exacerbations when added to inhaled therapy in severe asthma. However, there is little published evidence for effectiveness in patients treated with biologics. We conducted a retrospective audit of all patients who started azithromycin while on biologics in our centre. Compared with those that did not start azithromycin, these individuals had more exacerbations and a phenotype of chronic bronchitis and/or frequent purulent exacerbations. The addition of azithromycin to biologics was associated with reduced annual rates of steroid-treated and antibiotic-treated exacerbations and improved symptom scores (Asthma Control Questionnaire-5) but not with any improvement in lung function. Data support testing azithromycin in clinical trials in patients on biologics with residual exacerbations.

Exhaled nitric oxide (FeNO) and blood eosinophilia are important biomarkers of type-2 inflammation with additive predictive value in asthma. We have shown that high FeNO correlates with chemokines and cytokines involved in epithelial signalling whilst blood eosinophil levels correlate with serum interleukin-5 only.1 This suggests that FeNO more accurately reflects inflammation in the airway 'compartment' and blood eosinophil count reflects the systemic 'compartment' in eosinophilic asthma. We now test the hypothesis that the effect of low dose inhaled corticosteroids and oral corticosteroids, both effective treatments for eosinophilic airway inflammation, have differential effects on the airway (reflected by FeNO) and systemic compartment (reflected by blood eosinophils) in patients with eosinophilic asthma. Corticosteroid-naïve patients were assessed before and after 8 weeks of 200 mcg twice daily beclomethasone via aero-chamber. Patients with eosinophilic asthma established on inhaled treatment were assessed before and after 10 days of 30 mg prednisolone daily. Patients were at steady state and had evidence of type-2 airway inflammation reflected by either baseline FeNO >45ppb and/or blood eosinophils >0.30x109/L. 11 patients were recruited to the ICS group and 13 to the OCS group. Groups were matched for age but not baseline FEV1. Low-dose ICS produced a large median decrease in FeNO of 53.5%, p < 0.002, 95% CI [36.1, 71.0] and a small decrease in blood eosinophilia of 18.8% p < 0.005, 95% CI [6.5, 42.8] (figure 1). By contrast, OCS resulted in a comparatively smaller reduction in FeNO of 34.8%, p < 0.001, 95% CI [18.0, 51.5] but a large decrease in blood eosinophils of 80.3%, p < 0.001, 95% CI [64.3, 92.3]. These results suggest inhaled and oral steroids act differentially in the airway and blood compartments. The relative resistance to inhaled corticosteroids identified in some patients with type-2 high asthma, and the comparative success of oral therapy in this group, may reflect a greater contribution from the systemic reservoir of blood eosinophils. Reference Couillard S, Shrimanker R, Chaudhuri R, et al. Fractional Exhaled Nitric Oxide Non-suppression Identifies Corticosteroid-Resistant Type 2 Signaling in Severe Asthma. Am J Respir Crit Care Med. 2021;204(6):731–734.

To explore whether fractional exhaled nitric oxide (FeNO) non-suppression identifies corticosteroid resistance, we analysed inflammatory mediator changes during a FeNO suppression test with monitored high-intensity corticosteroid therapy. In linear mixed-effects models analysed over time, the 15 clinically distinct ‘suppressors’ (ie,≥42% FeNO suppression) normalised Asthma Control Questionnaire scores (mean±SD, start to end of test: 2.8±1.4 to 1.4±0.9, p<0.0001) and sputum eosinophil counts (median (IQR), start to end of test: 29% (6%–41%) to 1% (1%–5%), p=0.0003) while significantly decreasing sputum prostaglandin D2(254 (89–894) to 93 (49–209) pg/mL, p=0.004) and numerically decreasing other type-2 cytokine, chemokine and alarmin levels. In comparison, the 19 non-suppressors had persistent sputum eosinophilia (10% (1%–67%) despite high-intensity therapy) with raised end-test inflammatory mediator levels (1.9 (0.9–2.8)-fold greater than suppressors). FeNO non-suppression during monitored treatment implies biological corticosteroid resistance.

In T2‐mediated severe asthma, biologic therapies, such as mepolizumab, are increasingly used to control disease. Current biomarkers can indicate adequate suppression of T2 inflammation, but it is unclear whether they provide information about airway microbial composition. We investigated the relationships between current T2 biomarkers and microbial profiles, characteristics associated with a ProteobacteriaHIGH microbial profile and the effects of mepolizumab on airway ecology.

The human airways were once thought sterile in health. Now metagenomic techniques suggest bacteria may be present, but their role in asthma is not understood.

Background Clinical trials with mepolizumab, a humanised monoclonal antibody against interleukin-5, show a 50% reduction in severe asthma exacerbations in people with severe eosinophilic asthma. Exacerbations in patients treated with mepolizumab seem to be different to exacerbations in those given placebo, as patients treated with mepolizumab report fewer symptoms, have a lower sputum eosinophil count, and smaller fall in peak expiratory flow. We aimed to investigate the inflammatory phenotype and physiological characteristics of exacerbation events in patients with severe eosinophilic asthma who were treated with mepolizumab. Methods This multicentre, prospective, observational cohort study was carried out at four UK specialist severe asthma centres. Participants were aged 18–80 years, with severe eosinophilic asthma (Global Initiative for Asthma steps 4 and 5), and were eligible for mepolizumab therapy. All participants received mepolizumab 100 mg subcutaneously every 4 weeks, had a scheduled study visit when stable on mepolizumab (≥3 months on treatment), and measured daily peak flow and completed symptoms diaries throughout the course of the study. Participants attended their study centre for unscheduled exacerbation assessment when symptoms worsened outside of their normal daily variation and before commencing rescue treatment. If a participant was unable to attend their study centre for exacerbation or had initiated rescue treatment before the study visit, clinical details of the missed exacerbation were collected by clinical staff. In this exploratory study, the endpoint was 100 clinical assessments at exacerbation completed across all sites for participants on mepolizumab before initiation of rescue treatment. Characteristics of those who had exacerbations on mepolizumab were compared with those who did not, peak flow and symptoms diaries were compared for assessed versus missed exacerbations, and exacerbation phenotypes defined by sputum eosinophil cell count were compared. The utility of fractional exhaled nitric oxide (FeNO) and C-reactive protein in determining exacerbation phenotype on mepolizumab treatment were also assessed. This study is registered with ClinicalTrials.gov, NCT03324230. Findings Between Nov 30, 2017, and May 29, 2019, 145 participants were enrolled and treated with mepolizumab, five were excluded from the analysis. 172 exacerbations occurred, with 96 (56%) assessed before commencing rescue treatment. Compared with patients who did not exacerbate, patients who exacerbated had a higher exacerbation rate and more emergency department attendances in the year before commencing mepolizumab. The change in peak expiratory flow at nadir in the assessed exacerbation group was mean −40·5 L/min (SD 76·3) versus mean −37·0 L/min (93·0; p=0·84) in the missed exacerbation group, and there was no difference in reported symptom burden. When comparing exacerbations with a high sputum eosinophil count (≥2%; SEHIGH) with exacerbations with a low sputum eosinophil count (<2%; SELOW), the SEHIGH exacerbations were FeNO high (median difference 33 parts per billion [ppb; 95% CI 8 to 87]; p=0·0004), with lower FEV1 percent predicted (mean difference −15·9% [–27·0 to −4·8]; p=0·0075), lower FEV1 to forced vital capacity ratio (mean difference −10·3 [–17·0 to −3·6]; p=0·0043), and higher blood eosinophil counts (median difference 40 cells per μL [20 to 70]; p=0·0009). By contrast, SELOW exacerbations had higher C-reactive protein concentrations (median difference 12·7 mg/L [3·5 to 18·5]; p<0·0001), higher sputum neutrophil counts (median difference 52·7% [34·5 to 59·2]; p<0·0001), and were more likely to be treated with antibiotics (p=0·031). FeNO (≤20 or ≥50 ppb) was the most useful discriminator of inflammatory phenotype at exacerbation. The most common adverse event was hospital admission due to asthma exacerbation (17 [50%] of 34 events), none of the adverse events were study procedure related. Interpretation Exacerbations on mepolizumab are two distinct entities, which can largely be differentiated using FeNO: non-eosinophilic events are driven by infection with a low FeNO and high C-reactive protein concentration, whereas eosinophilic exacerbations are FeNO high. The results of the MEX study challenge the routine use of oral corticosteroids for the treatment of all asthma exacerbation events on mepolizumab, as well as the switching of biological therapies for treatment failure without profiling the inflammatory phenotype of ongoing asthma exacerbations. The results highlight clinically available tools to enable profiling of these residual exacerbations in patients treated with mepolizumab. Funding UK Medical Research council.