Single-cell RNA-sequencing of circulating eosinophils from asthma patients reveals an inflammatory signature

Abstract

Asthma is the most common chronic lung disorder in the United States. While asthma is heterogeneous, blood eosinophils are central to the pathogenesis in most cases. Yet, the power of modern omics has not been widely applied to the study of asthma eosinophils. We report single cell RNA sequencing of blood eosinophils obtained from patients with severe asthma, mild asthma, and healthy volunteers. The eosinophils from asthma patients showed marked heterogeneity in the population and, as with healthy controls, clustered into 3 subsets suggesting at least 3 gene expression states circulating in the blood. Eosinophils from asthma patients had an inflammatory gene signature with enrichment of interferon α and γ pathways. Moreover, a greater fraction of these eosinophils expressed CCR3, the chemokine receptor that mediates trafficking to inflamed tissues and activates eosinophils. Our data support implementation of larger studies to define the transcriptional drivers of asthma.

Keywords: Health sciences; Immunology; Medical specialty; Medicine.

Graphical abstract

Figure 1

Healthy human eosinophils have a gene expression profile distinct from other MNCs and granulocytes (A) Experimental schema to obtain scRNA-seq of asthmatic and healthy eosinophil and mononuclear cells. (B) UMAP plot of healthy eosinophils, neutrophils, and mononuclear cells (n = 51,400). (C) Heatmap of averaged expression of top 500 most significant healthy eosinophil marker genes (Padj <0.05, log2FC > 0.25). Scale represents gene expression Z score. (D) Violin plots of gene expression for canonical eosinophil markers. Adjusted p-values from differential expression testing (Wilcoxon rank-sum test). (E) Heatmap of averaged expression of 154 degranulation pathway genes. Scale represents gene expression Z score.

Figure 2

Healthy eosinophils are heterogeneous and vary in expression of essential effector genes (A) UMAP of all eosinophils (n = 24,538) colored by patient (left) condition (middle) and cluster (right). (B) UMAP of healthy eosinophils only (left) and bar plot of the fraction of healthy cells per cluster (right). (C) Heatmap of average expression of cluster markers (Padj <0.05, log2FC > 0.25). Scale represents gene expression Z score. (D) Top gene ontology biological process pathways enriched in each cluster (Padj <0.05). (E) Violin plots of expression of primary and secondary granule genes and HLA genes. p values were determined by the Kruskal-Wallace test. (F) Dot plot of top interferon-stimulated gene markers for cluster 3 colored by scaled average expression. Adjusted p value from Wilcoxon rank-sum test.

Figure 3

Asthmatic eosinophils have distinct gene expression profiles from healthy eosinophils (A) UMAP plot of down sampled eosinophils from healthy and asthma patients colored by condition (left) and cluster (right). (B) Bar plot of fraction of cells per condition for each cluster. (C) Volcano plot of significant DEGs in healthy vs. asthmatic eosinophils (P_adj < 0.05, log2FC > 0.25). The top 5 most significant genes per group are labeled. (D) Violin plots of expression of class I HLA genes, antigen presentation genes, granule genes and CCR3. Adjusted p value from Wilcoxon rank-sum test.

Figure 4

Asthmatic eosinophils exhibit an inflammatory gene signature (A) GSEA line plots of enriched hallmark pathways in asthma compared to healthy: hallmark interferon gamma response (left), hallmark interferon alpha response (middle), and hallmark inflammatory response (right). (B) Cord plot of 13 genes in the hallmark interferon gamma response, hallmark interferon alpha response, and hallmark inflammatory response pathways which are significantly overexpressed in asthmatic eosinophils compared to healthy.

Figure 5

Mild and severe asthmatic eosinophils have distinct gene expression profiles (A) UMAP clustering of all eosinophils colored by disease severity. (B) Bar plot of fraction of cells per condition for each cluster. (C) Volcano plot of significant DEGs between mild and severe asthmatic eosinophils (P_adj < 0.05, log2FC > 0.25). The top 5 most significant genes per group are labeled.

Figure 6

Severe asthmatic eosinophils exhibit an inflammatory gene signature (A) Normalized enrichment scores (NES) from GSEA analysis of hallmark gene sets. A negative NES indicates the pathway is enriched in severe eosinophils, and a positive NES indicates the pathway is enriched in mild eosinophils. (B) GSEA line plot (left) and UMAP plot colored by activity score of enriched hallmark interferon alpha response genes (right). (C) GSEA line plot (left) and UMAP plot colored by activity score of enriched hallmark interferon gamma response genes. (D) Selected interferon-stimulated genes which are significantly overrepresented in severe asthmatic eosinophils compared to mild.