D21-18 Linking Airway Physiology and Inflammatory Endotypes in Asthma to Airway Leukocyte Profiles at Single-Cell Resolution

Rationale Indirect airway hyperresponsiveness (AHR) is a specific feature of airway dysfunction in asthma but the immunopathological mechanisms responsible for indirect AHR remain incompletely understood. We have previously characterized transcriptional circuits implicated in indirect AHR, which involved signaling between the airway epithelium, mast cells (MCs), and eosinophils, through bulk RNA-sequencing analysis of epithelial brushings (Murphy AJRCCM 2023). Here we examine leukocyte populations in induced sputum samples obtained from individuals with and without asthma who underwent extensive phenotyping for AHR using fixed single-cell RNA sequencing (scRNA-seq). Methods Induced sputum was collected from 3 healthy individuals and 8 individuals with asthma not receiving immune-modulating therapies who underwent characterization for direct (methacholine challenge) and indirect AHR (mannitol challenge) and T2 endotype. After squamous epithelial cell removal, samples were fixed and scRNA-seq was performed using the 10X Chromium Fixed RNA Profiling platform. Data were analyzed using Seurat v5 for filtering, integration, clustering, & dimensionality reduction. Pseudobulk samples were used for differential gene expression analysis using DESeq2. Results 17,474 genes were quantified across 96,083 cells (average of 8,735±3,252 cells/sample). Five major leukocyte populations were identified by canonical markers (Fig. 1A-B), including neutrophils. We could not identify a definite eosinophil population. Proportions of cellular subsets did not significantly differ between asthma phenotype or presence or absence of indirect AHR (Fig. 1C) except for the MC/basophil cluster, which was identified in higher proportions in individuals with T2-high asthma and individuals with indirect AHR (Fig. 1F). Subclustering of mononuclear phagocytes revealed seven distinct subpopulations, including three macrophage clusters (Fig. 1D). We also identified seven subpopulations in the non-B-cell lymphocyte cluster, including ILC2s (Fig. 1E). Differential expression analysis within the neutrophil cluster identified 128 differentially expressed genes (FDR <0.10) between cells from T2-high versus non-T2 asthma, including higher expression of ALOX5, IL4R, and CISH in neutrophils from T2-high asthma and higher expression of C3, CXCR4, CXCL8, CD63, and SLPI in neutrophils from non-T2 asthma. Conclusions Sample fixation prior to scRNA-seq allows for successful capture of leukocyte populations from induced sputum samples in asthma, including neutrophils and other rare immune populations (mast cells/basophils, ILC2s). Preliminary results identify higher numbers of MCs/basophils in the airway lumen of individuals with T2-high asthma and those with indirect AHR, consistent with prior work. We also identified notable T2 endotype-specific differences in the transcriptional profile of airway neutrophils, which provide novel insights into the potential roles of neutrophils in both T2-high and non-T2 asthma. This abstract is funded by: NIH, Parker B.