Abstract Text: Mononuclear phagocytes (MNP) regulate tissue homeostasis and inflammation but are not well-characterized in human airways during allergic inflammation. We leveraged segmental allergen challenge (SAC) in allergic asthmatics (AA) and allergic non-asthmatic controls (AC) to distinguish transcriptional profiles specific to asthma from those associated with allergy alone. Single-cell RNA-sequencing was performed on endobronchial brushings collected at baseline and 24 hours after SAC (AC=4; AA=4), and on co-cultures of blood CD14+ monocytes and primary airway epithelial cells (AEC) at air-liquid interface. Logistic regression identified cells associated with AC (OR < 1) or AA (OR>1). Differential gene expression analysis was performed between groups (FDR < 0.1). Bronchoalveolar lavage protein was quantified via multiplex assay (AC=14; AA=13). Clustering of 8,510 airway mucosal MNP identified 14 subsets including macrophages, dendritic cells, and monocyte-derived cells (MC). MC2 (SPP1) associated with AC at baseline (OR 0.52, [0.31-0.89]) and after SAC (OR 0.26, [0.18-0.36]). MC4 (CCR2) associated with AA after SAC (OR 2.77 [1.29-5.92]) and upregulated IL-13-induced genes involved in eicosanoid synthesis and matrix remodeling, including MMP12. MMP-12 protein levels were higher in AA compared to AC after SAC (745.7 [176.7-1957.7] vs. 228.6 [69.7-493.5] pg/mL, P=0.02). RNA velocity predicted a differentiation continuum from MC4 to MC2, with acquisition of genes directing macrophage identity, phagocytosis, and lipid metabolism. Consistent with this, blood monocytes transcriptionally aligned with MC4 but acquired an MC2 profile after co-culture with AEC. Thus, type 2 cytokine signaling in asthmatic airways may prevent macrophage differentiation and instead arrest MC in a pathogenic state that drives airway inflammation and pathologic remodeling.
