Anti-inflammatory effects of oral prednisolone at stable state in people treated with mepolizumab: a proteomic and bulk transcriptomics analysis

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 Statement IH 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 Trial This study is registered on Clinicaltrials.gov ([NCT03610685][1]). ### Funding Statement This 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 Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The 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. Yes I 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). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Anonymised 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. 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