ABSTRACT: Type-2 (T2) cytokine-driven eosinophilic exacerbations in severe asthma respond to targeted biological therapies, but the mechanisms of residual disease expression when T2-cytokine biology is suppressed are poorly understood. Pathological studies indicate that ongoing mast cell activation and persistent airway remodelling are important factors contributing to symptoms, reduced lung function, and non T2 disease. The molecular mechanisms driving these features of asthma in the absence of T2 biology are ill-defined. To explore the molecular pathways that are dysregulated in T2 biomarker-high and -low severe asthma. T2 biomarker-high severe asthma (T2-high, n=18) was compared to biomarker-intermediate (T2-intermediate, n=23) and biomarker-low (T2-low, n=11) severe asthma, and healthy controls (n=20). Bronchoscopy samples were analysed by bulk RNAseq. Treatment-optimised asthma, independent of confounding by therapeutic corticosteroids, was characterised by upregulation of the mucins MUC5AC, MUC2, lysozyme, CEACAM5, SYNCRIP, typical T2-genes POSTN and IL33, the transcription factor FOS; and ITGB8 which regulates TGF-β activation and airway remodelling. When comparing between phenotypes steroid-resistant T2-high severe asthma was characterised by upregulation of expected T2 high genes, epithelial barrier genes and keratins. T2-low asthma was characterised rather by Th1 and Th17 associated genes, including IDO1, CXCL10, GBP1, the T cell checkpoint receptor LAG3, neuroimmune genes SGC2 and CALCA, and by enrichment of mast cells. Steroid-responsive T2-intermediate asthma was distinctly characterised by expression of the antimicrobial peptide BPFIA1, virus activated signatures, enrichment for mast and NK cells, and distinct transcription factors NFKB1, TBX21, IRF1, IRF2, BATF and STAT2 associated with pathogen defence. We have characterised three major asthma phenotypes, independent of confounding by therapeutic corticosteroids, identifying gene sets and pathways distinct to each.
