Characterization of the Bronchoalveolar Lavage Fluid by Single Cell Gene Expression Analysis in Healthy Dogs: A Promising Technique

Abstract

Single-cell mRNA-sequencing (scRNA-seq) is a technique which enables unbiased, high throughput and high-resolution transcriptomic analysis of the heterogeneity of cells within a population. This recent technique has been described in humans, mice and other species in various conditions to cluster cells in populations and identify new subpopulations, as well as to study the gene expression of cells in various tissues, conditions and origins. In dogs, a species for which markers of cell populations are often limiting, scRNA-seq presents with elevated yet untested potential for the study of tissue composition. As a proof of principle, we used scRNA-seq to identify cellular populations of the bronchoalveolar lavage fluid (BALF) in healthy dogs (n = 4). A total of 5,710 cells were obtained and analyzed by scRNA-seq. Fourteen distinct clusters of cells were identified, further identified as macrophages/monocytes (4 clusters), T cells (2 clusters) and B cells (1 cluster), neutrophils (1 cluster), mast cells (1 cluster), mature or immature dendritic cells (1 cluster each), ciliated or non-ciliated epithelial cells (1 cluster each) and cycling cells (1 cluster). We used for the first time in dogs the scRNA-seq to investigate cellular subpopulations of the BALF of dog. This study hence expands our knowledge on dog lung immune cell populations, paves the way for the investigation at single-cell level of lower respiratory diseases in dogs, and establishes that scRNA-seq is a powerful tool for the study of dog tissue composition.

Keywords: bronchoalveolar lavage fluid; cell; dog; lung; single-cell RNA-sequencing.

Figure 1

Compiled t-SNE plot of the cell clusters. (A) t-SNE plot of all cells (n = 5,710) representing the cell clusters analyzed by scRNA-seq. Each color corresponds to one cluster assigned via the graph-based clustering method with a resolution of 0.3. (B) Batch alignment across bronchoalveolar lavage fluid (BALF) specimens, each color representing the cells coming from one sample. (C) Bar plot showing the relative proportion of the cell from each BALF sample into each cluster. BALF 1, female Yorkshire terrier of 11-year-old; BALF 2, female French bulldog of 4-year-old; BALF 3, female West Highland white terrier of 9-year-old; BALF 4, female Australian shepherd of 6-year-old.

Figure 2

Identification of cell identity corresponding to the clusters. t-SNE plot showing the cells identity based on the expression of differentially expressed genes representative of each cells type including genes coding for the macrophage receptor with collagenous structure (MARCO), the macrophage mannose receptor (MRC1, encoding CD206), the T-cell surface glycoprotein CD3 epsilon chain (CD3E), the cytokeratin 19 (KRT19), the selectin (SELL, encoding CD62L), the integrin alpha M (ITGAM), the T-cell surface glycoprotein CD1e (CD1E), the CD83 molecule, the Fc receptor like A (FCRLA), the CD19 molecule, the DNA topoisomerase II alpha (TOP2A), the proliferating cell nuclear antigen clamp associated factor (ENSCAFG00000030087, encoding PCLAF), the membrane spanning 4-domains A2 (MS4A2) and the mast/stem cell growth factor receptor (KIT). DC, dendritic cell.

Figure 3

Single-cell mRNA-sequencing based identification of 4 distinct subpopulations of macrophages/monocytes in the bronchoalveolar lavage fluid of dogs. Dot plots showing the average expression of the indicated genes as well as the percentage of cells expressing the genes within each cluster of macrophages/monocytes. An example of transcripts significantly (P-value adjusted < 0.05) differentially upregulated (average log2 fold change > 0.5) between the clusters 0, 3, 5, and 8 are depicted.