D21-15 Altered Epithelial Repair and Dysregulated Epithelial Cell Activity in Severe Asthma as Revealed by Single-cell RNA-seq

Abstract Rationale Airway epithelial remodeling, characterized by goblet-cell expansion and potential loss of ciliated cells, is a defining feature of severe asthma (SA). However, the transcriptional mechanisms that initiate or sustain this remodeling process remain incompletely understood. We aimed to delineate airway epithelial cell alterations associated with SA at single-cell resolution and identify early events underlying epithelial remodeling. Methods Bronchial brush samples were collected from 14 individuals with SA and 8 healthy controls (HCs) encompassing discovery and validation cohorts. SA participants were older on average and all were receiving inhaled corticosteroids, with 43 percent also treated with biologic therapies. After quality control, single-cell RNA sequencing yielded transcriptomes for 127,437 single cells. Datasets were integrated and clustered using Seurat1, and epithelial subsets were annotated based on well-established markers2 and prior knowledge. Differential expression and pathway enrichment analyses were performed to identify cell-type-specific dysregulation in SA compared with HC. Results The integrated dataset consistently captured both major and rare epithelial and immune populations across cohorts. SA epithelia showed coordinated up-regulation of metabolic, redox, and cellular stress-response programs, along with down-regulation of cilium-assembly and axonemal-organization genes, indicating disrupted epithelial homeostasis. This breakdown of homeostasis was marked by the emergence of airway remodeling signatures, beginning with the basal cells. In HCs, resting basal cells maintained a cilia-directed pre-assembly transcriptional state, expressing genes involved in ciliogenesis and axonemal assembly. In SA, resting basal cells instead adopted an interleukin-13-skewed abnormal-repair program marked by increased expression of early stress-response genes (FOS, JUN) and IL-13-responsive secretory and mucus-associated genes (ALOX15, MUC5AC, AGR2, TFF3) in the relative absence of cilia-promoting transcripts. Other epithelial subsets, including secretory and ciliated cells, exhibited enrichment of pathways related to cellular stress, innate immune activation, and impaired differentiation. Conclusions Single-cell transcriptomic profiling revealed that in severe asthma, basal cells exhibited early transcriptional changes indicating reduced ciliated-lineage commitment and enhanced secretory differentiation. These findings support the possibility of a stress-induced divergence of cell differentiation states at the early (basal cell) stage which may drive the abnormal mucus generating epithelium associated with asthma. UMAP visualization of epithelial and immune cell types from pooled bronchoscopy samples of 8 healthy controls (HCs) and 14 severe asthma (SA) patients. References 1. Hao, Y. et al. Integrated analysis of multimodal single-cell data. Cell 184, 3573-3587 e3529 (2021). 2. Goldfarbmuren, K.C. et al. Dissecting the cellular specificity of smoking effects and reconstructing lineages in the human airway epithelium. Nat Commun 11, 2485 (2020). This abstract is funded by: NIAID