D21-07 The Asthmatic Airway Niche Imprints Functional Neutrophil Programs

Abstract Rationale Asthma spans eosinophilic T2 and neutrophilic T1 airway endotypes. While T2-high asthma has effective biologics, mixed eosinophilic/neutrophilic and neutrophil-predominant endotypes are more severe and treatment-resistant. Despite their implication in pathobiology, how neutrophils sustain airway inflammation and dysfunction remains poorly defined. We hypothesized that (i) epithelial-neutrophil crosstalk and (ii) T1 vs T2 cytokine exposure independently program distinct neutrophil states that drive disease. We used complementary systems: (1) a primary airway epithelial-neutrophil co-culture and (2) matched neutrophil cytokine-stimulation monocultures, to define how epithelial conditioning and T1/T2 niches imprint neutrophil activation and function. Methods Primary human bronchial epithelial cells were differentiated at air-liquid interface (ALI) for 21 days to generate mucociliary cultures. Purified blood neutrophils were added basolaterally to ALI Transwells (1 × 10⁶ cells/well) or maintained in monoculture. Monoculture neutrophils were stimulated 48 hours with vehicle, IL-13 (10 ng/mL; T2), or IFN-γ (10 ng/mL; T1) ; in parallel, neutrophil-epithelium co-cultures were maintained for 48 hours without exogenous cytokines. Neutrophils from all conditions were harvested for functional assays and bulk RNA-seq. Analyses focused on (i) unstimulated neutrophil monoculture vs epithelial co-culture and (ii) neutrophil cytokine-stimulated vs vehicle monoculture. Results Versus monoculture, epithelial co-culture induced an amoeboid morphology consistent with enhanced migration and altered the expression of 4, 002 genes in neutrophils. Upregulated gene programs encompassed PAMP sensing (TLR1/2/4/6/10), NF-κB/JAK-STAT signaling (NFKB1/2/IZ, RIPK2, TNFAIP2/3/6), chemokine pathways (CXCL1/8/16), metabolic rewiring (SLC2A3/14, HIF1A, NAMPT) and adhesion/motility (ITGAM, ITGAX, ICAM1). IFN-γ stimulation induced wide-scale reprogramming of neutrophils with 3, 405 differentially expressed genes (DEGs) identified. Top upregulated genes reflected intense IFN signaling (STAT1, IRF1/2/5/9, IFIT1/2/3/5), activation of antigen processing machinery (TAP1/2, PSMB8/9), and chemokine signaling (CXCL8, CCL2, CCR1). IL-13 stimulation induced much fewer, but still significant transcriptional changes (DEGs=304) including upregulation of the eosinophil chemotactic factor CCL24, lectin/glycan recognition (CLEC10A, CD209), and lipid/GPCR chemo-sensing genes (HCAR2/3, OXER1, SUCNR1), alongside downregulation of neutrophil IL-8 axis and activation receptors (CXCR1/2, CSF3R, FCGR3A/B). Conclusions Epithelial conditioning yields neutrophils with enhanced migratory potential, activation, and surveillance programs. IFN-γ directs activation of a broad antiviral/antigen-processing state, whereas IL-13 rewires chemosensing toward glycans/lipids and away from IL-8/CXCR1/2 trafficking cues. Thus, the airway niche, epithelium and asthmatic cytokines, imprint discrete, functionally significant neutrophil programs that can propagate neutrophilic airway pathology and suggest novel therapeutic targets. This abstract is funded by: 1U19AI181102