D21-14 Examining Human Sputum Neutrophil Differential Gene Expression Associated With Asthma Biologics

Abstract Rationale Biologics directed against the Type 2 inflammatory cytokines IL-5, IL-4, and IL-13, or their receptors, are increasingly used to treat moderate-to-severe asthma. Eosinophils and neutrophils are key effector cells in asthma, but the transcriptomic effects of these medications on these cells are not fully defined, particularly at single cell resolution. Moreover, there is mounting evidence that eosinophils and neutrophils can influence one another in the airway and that IL-5 may directly influence neutrophils. Therefore, we investigated the effect of IL-5 receptor blockade by benralizumab on neutrophil gene expression in sputum neutrophils isolated from donors with asthma. Methods We isolated live CD45+ immune cells in sputum from 11 donors with asthma and performed single-cell RNA sequencing (10X Genomics). We annotated ∼123,000 neutrophil single cell transcriptomes in this dataset (Genes/cell 200, mitochondrial transcripts 10%) and performed differential gene expression analysis (DGEA) comparing neutrophils from donors treated with benralizumab (n = 2) to donors not treated with any asthma biologic (n = 7), employing a Wilcoxon method with significance defined as FDR 0.05 and Log2 Fold change ≤ -0.5 or ≥ 0.5. We next performed gene set enrichment analysis (GSEA). For perspective, we also compared neutrophils from donors treated with IL-4 receptor antagonist dupilumab (n = 2) to those from the untreated donors. Results There were 690 differentially expressed genes (DEG) in the benralizumab group and 778 in the dupilumab group, each compared to the untreated group (Figure). Notable DEG in the benralizumab group included IL18R1 and IRAK3 (upregulated), and IL8 and FCGR3b (downregulated). GSEA demonstrated downregulation of genes related to “Interferon signaling” and “Positive Regulation of IL-6 Production. In the dupilumab group, notable DEG included IFITM2 and HLA-C (upregulated) and APOC1 and PTPRC (downregulated). Meanwhile, upregulated genes were enriched for processes including “antigen presentation” and “interferon α/β signaling”. Conclusions Biologic therapies have transformed the management of severe asthma, yet their effects on granulocytes at the cellular level remain to be fully determined. This preliminary analysis suggests a significant effect of IL-5 receptor blockade on neutrophil gene expression, one partly characterized by dampened expression of interferon related genes. This could be a direct effect, or one mediated by loss of intercellular crosstalk due to depletion of airway eosinophils. Meanwhile, dupilumab is associated with increased antigen presentation and interferon signaling-related genes. Larger prospective studies comparing granulocytes from patients before and after asthma biologic therapy will help fully contextualize these preliminary findings. This abstract is funded by: NIH