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.

Background Mepolizumab and prednisolone have overlapping anti-inflammatory effects so the clinical effects of prednisolone might be attenuated in severe eosinophilic asthma (SEA) patients treated with mepolizumab. Methods We tested this hypothesis in a randomized, double-blinded, placebo-controlled, crossover trial of prednisolone (0.5 mg/kg/day, 2 weeks) after ≥12 weeks of mepolizumab. Symptoms and quality of life (QoL) questionnaire scores, lung function including oscillometry and markers of inflammation were measured before and after prednisolone and placebo. Results There were no significant changes in asthma symptoms and QoL questionnaire scores following prednisolone treatment. In comparison to placebo, prednisolone improved FEV1 by 100 ml (p=0.019) and FEF25-75 by 200 ml/s (p=0.006). Median FeNO at baseline was 37ppb. Prednisolone reduced FeNO by 13.0ppb (p=0.001), blood eosinophil count by 0.02 × 109/L (p=0.003) and sputum eosinophil percentage of total cell count by 1.4% (p=0.002) in comparison to placebo. Post-prednisolone and post-placebo SNOT-20 questionnaire scores indicated there were no improvements in nasal symptoms following prednisolone in patients on mepolizumab. Conclusion In patients with severe eosinophilic asthma treated with mepolizumab, prednisolone has no significant effects on symptoms or quality of life but improves FEV1 and small airway function and reduces FeNO.

Background Culture independent techniques demonstrate altered airway ecology in severe asthma at stable and exacerbation states. Biological cluster analysis across severe asthma and COPD has shown distinct cytokine and microbiological profiles,1 suggesting that certain phenotypes may be more responsive to antimicrobial therapies. Previous work in COPD suggests that measuring the relative proportions of 2 dominant bacterial phyla, Gammaproteobacteria (G) and Firmicutes (F), or G:F ratio, may provide a useful biomarker to identify these groups.2 Objective To characterise subjects with stable severe asthma separated by cluster analysis according to airway microbiology. Methods Patients with severe asthma were prospectively recruited to a single UK centre. Sputum samples were obtained from 63 clinically stable patients for microbiome analysis. Hierarchical cluster analysis according to phylum was performed and clinical characteristics compared between groups. Results Three microbiological clusters were identified. Cluster 1 had a high G:F ratio and sputum microbial communities were dominated by the potentially pathogenic organisms Haemophilus and Moraxella (76% total pathogens). Clusters 2 and 3 had lower proportions of these organisms (23% and 7% respectively) with equivalent and low G:F ratios. Whilst no statistically significant difference in clinical characteristics was observed between groups, there was a trend towards increased bacterial load in cluster 1 (5.69 × 109 CFU/ml) compared to clusters 2 and 3 (1.41 and 2.84 × 109 CFU/ml, p=0.067). The high G:F group did not have a higher total neutrophil count or% neutrophil count as compared to other groups. There was no significant difference between clusters in exacerbations/year at baseline, as defined by either oral corticosteroid or antibiotic requirement. Conclusion Microbiological clustering in severe asthma identifies three groups with significantly different G:F ratios. Further testing is required to ascertain whether this measure is a useful biomarker, for example, to identify those likely to respond to maintenance antibiotic therapy. References Ghebre MA, Pang PH, Diver S, et al. Biological exacerbation clusters demonstrate asthma and chronic obstructive pulmonary disease overlap with distinct mediator and microbiome profiles. J Allergy Clin Immunol2018;141(6):2027–36 e12. Haldar K, Bafadhel M, Lau K, et al. Microbiome balance in sputum determined by PCR stratifies COPD exacerbations and shows potential for selective use of antibiotics. PLoS One2017;12(8):e0182833.

Asthma is a common chronic inflammatory condition of the airways affecting over 300 million people world-wide. In 5%-10% of cases, it is severe, with disproportionate healthcare resource utilization including costs associated with frequent exacerbations and the long-term health effects of systemic steroids. Characterization of inflammatory pathways in severe asthma has led to the development of targeted biological and small molecule therapies which aim to achieve disease control while minimizing corticosteroid-associated morbidity. Herein, we review currently licensed agents and those in development, and speculate how drug therapy for severe asthma might evolve and impact on clinical outcomes in the near future.