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Rationale: Activation of multiple cytokine receptors is reported to play an important role in asthma via the Janus kinases (JAK) pathway. IL-13 is known to activate JAKs. We investigated whether this pathway was activated in the sputum and blood of patients from the U-BIOPRED cohort. Methods: Sputum transcriptomics data from 120 subjects with severe asthma (SA) who were smokers (SAsm) or non-smokers (SAn), mild moderate asthmatics (MMA) or healthy controls (HC). Blood transcriptomic data from 312 subjects was also obtained. We used gene set variation analysis (GSVA) to study the KEGG JAK-STAT signalling pathway. IL-13 expression in sputum was detected by SomaScan. Results: The JAK-STAT signalling pathway was enriched in the sputum of asthmatic subjects compared to HC although this was only significant in the SAn group (p=0.01). Subgrouping asthmatics according to their sputum granulocyte status indicated that patients with neutrophilic and mixed granulocytic asthma had the highest JAK-STAT enrichment compared to HC, eosinophilic asthma and paucigranulocytic asthma (all FDR Conclusions: High activation of JAK-STAT signalling in neutrophilic and mixed granulocytic asthma suggests that these subjects may be suitable for inhaled anti-JAK therapy.

We hypothesized that Gene Set Variation Analysis (GSVA) of gene expression in bronchial brushings can effectively classify airway Th2-high status in U-BIOPRED asthmatics. Airway Th2-status was defined as IL-13-induced transcriptional activity, evaluated as enrichment of IL-13 in-vitro stimulation (IVS) or expression of POSTN and SERPINB2 in human bronchial epithelial cell transcriptomic profiles from U-BIOPRED asthma patients. Based on IL-13 IVS GSVA enrichment scores above the 95 th percentile for healthy subjects, 53% of severe asthma (SA) non-smokers (n=49), 67% of SA smokers (n=18) and 36% of non-severe asthmatics (n=36) were classified as Th2-high. Eosinophilic inflammation marker values are shown in Figure 1. Using the mean normalised expression of POSTN and SERPINB2 as an alternative Th2 stratification factor, 35% of SA non-smokers, 22% of SA smokers, and 14% of non-severe asthmatics were Th2-high, mostly subsets of the IL-13 IVS-defined Th2-high asthma groups and consistent with the modest correlation (r=0.39) between the IL-13 IVS enrichment scores and POSTN/SERPINB2 scores. IL-13 IVS and POSTN/SERPINB2 based definitions of Th2 activity showed associations with eosinophilic and atopy markers, with good agreement for Th2-high vs. -low calls, although the latter approach may be too conservative. IL-13 IVS enrichment identified a broader set of subjects with airway Th2 activity when compared to POSTN and SERPINB2. Funded by IMI.

Cell proliferation and intracellular oxidative stress are enhanced in asthma. Mitochondria are associated with these processes. Regulation of mitochondrial function therefore provides a novel target in understanding disease mechanisms. We investigated mitochondrial function through changes in activity, membrane potential, reactive oxygen species production and pro-inflammatory cytokine release in airway smooth muscle cells (ASMCs) isolated from healthy control, non-severe and severe asthmatic patients, at baseline and following treatment with hyperproliferative stimuli (TGFβ and FBS) and oxidative stress (H 2 O 2 ) over 4, 24 and 48 hours. Mitochondrial function was altered in response to TGFβ+FBS and to H 2 O 2 , with an accumulation of mitochondrial reactive oxygen species (Fig 1), disruption to the mitochondrial membrane potential (ΔΨm, significantly decreased at 4 hours after TGFβ+FBS in all patient types, p These data suggest that an intrinsic ASMC mitochondrial dysfunction per se is not driving the asthmatic phenotype but is a secondary response to the inflammatory/proliferative environment in the lung.

Background: Severe asthma is characterised by poor therapeutic response to corticosteroids and a diverse airway inflammation profile. We have shown that inflammasome-associated genes are highly differentially expressed in sputum in the U-BIOPRED adult severe asthma (SA) cohort. Aims & Objectives: To investigate mediator profiles in sputum supernatants from subjects in U-BIOPRED. Methods: 1129 analytes were measured in sputum supernatants (Somalogic SOMAscan TM V3), with 111 samples passing quality control (with squamous cell content Results: 54 analytes were differentially expressed between SA non-smokers (n=43) and healthy subjects (n=18) (FDR 2-fold /healthy cohort). Pregnancy-associated plasma protein A (PAPP-A), serum amyloid P (SAP) and C-reactive protein (CRP) were highly up-regulated in both smoking and non-smoking SA (14.7-, 6.27- and 3.21-fold). PAPP-A (r=087;p -10 ), SAP (r=0.6;p -10 ) and periostin (r=0.55;p -10 ) correlated with sputum eosinophils, while b-endorphin (r=0.67;p -10 ), azurocidin (r=0.63;p -10 ), IL-5 (r=0.6;p -10 ) and IL-8 (r=0.51;p -8 ) with sputum neutrophils. These data are consistent with transciptomic data showing up-regulation of IL-1 receptor family gene transcripts, which are upstream of IL-6/acute phase response signalling, in SA. A similar profile of mediators has been seen in the Airways Disease Endotyping for Personalised Therapeutics (ADEPT) study. Conclusion: Severe asthma is associated with acute phase and eosinophil- and neutrophil- associated analytes in sputum. Inflammasome activation appears strongest in eosinophilic asthma.