Abstract Rationale Severe eosinophilic asthma (SEA) is associated with high numbers of eosinophils in blood and sputum. Cytokines IL-5 and IL-3 involved in the maturation, activation and release of eosinophils into the circulation, are elevated in SEA serum. Mepolizumab, an anti-IL5 therapy used in SEA removes 80% circulating blood eosinophils. However, the biology of the remaining eosinophils, particularly in relation to their gene and protein expression, is poorly explored. Aims Determine gene and protein expression profiles, and biologic response of eosinophils from SEAs established on the Mepolizumab (mSEA), SEAs not receiving anti-IL5 Mepolizumab (SEA) with and without cytokine stimulation. Methods Eosinophils were isolated from whole blood via Percoll density centrifugation and magnetic negative selection from SEA patients established on mepolizumab therapy (mSEA; n = 10) and SEAs (n = 12). RNAseq (n = 7; DESeq2 analysis) and O-Link Inflammation Panel (n = 6; normalised protein expression analysis) was performed on eosinophils and associated supernatant from unstimulated and after 24-hours of IL-5 or IL-3 stimulation in culture. sPLS was used to identify RNA and protein features and their key associations. Group-specific RNA-protein (RP) networks were built from correlations, retaining only edges that remained stable under leave-one-out resampling. Cell survival was measured using Annexin V stain (n = 5). Results The multi-omic network analysis comparing IL-5 stimulated and unstimulated SEA eosinophils identified 15 stable negatively correlated RNA-protein edges. In IL-5 stimulated SEA eosinophils, IL-13, IL-2RB and IL-7 were identified as hub proteins connected to transcripts including GSTM4, IPO8 and VPS33B. In contrast, mSEA IL-5 stimulated eosinophils, identified 0 unique edges with a loss of CXCL1, CXCL9 and CXCL10 chemokine/receptor modules when compared to unstimulated mSEA eosinophils. Correspondingly, 0 genes were increased and 3 genes decreased with IL-5 stimulation in mSEAs eosinophils. However, with IL-3 stimulation sPLS analysis identified 15 RNAs and proteins with 95 edges unique to the mSEA IL-3 stimulated eosinophils compared to unstimulated. Hub proteins AXIN1, CASP-8, OSM, TNF and SIRT2 involved in eosinophil recruitment, ECM remodelling and cell survival were identified with IL-3 stimulation not seen in the mSEA unstimulated eosinophils. Functional cell survival assays identified significantly increased cell survival with IL-3 (p = 0.047) but not with IL-5 stimulation in the mSEA eosinophils. Conclusions Eosinophils from patients on Mepolizumab therapy display a reduced capacity to respond to IL-5 stimulation, with no new networks or RP correlations identified. The increased response to IL-3 suggests these eosinophils are less primed to respond to IL-5 during maturation, identifying a potential distinct activation profile. This abstract is funded by: GSK supported study: ISS# 10825; Study# 213665
Raby et al. (Fri,) studied this question.