Characterization of the bronchoalveolar lavage fluid by single cell gene expression analysis in healthy dogs: a promising technique.

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).

Cells in suspension isolated from fresh BALF were loaded into the ChromiumTM and were directly encapsulated with unique barcoded primers using the drop‐sequencing method. Cells were lysed and reverse transcription of mRNAs took place into vesicles. cDNA was amplified after the breakage of the vesicles and sequenced on an Illumina NextSeq500. The analysis of the results and statistical analysis were performed using Cell Ranger software (v1.2.0), Seurat package in RStudio (v3.1.2) and the gene set enrichment analysis tool (GSEA‐P).

A total of 5710 cells were obtained and analyzed. Fourteen distinct clusters of cells were identified, further identified as alveolar macrophages (AMs) (3 clusters) and monocytes‐derived macrophages (1 cluster). The first cluster of AMs composed by the majority of cells exerted functions involved in immune defense and response, the second cluster exerted functions involved in immune response regulation and the third exerted functions involved in metal ions homeostasis. Clusters of CD8+ and CD4+ T cells were also found (1 cluster each) as well as clusters of mature and immature dendritic cells (1 cluster each) and clusters of ciliated or non‐ciliated epithelial cells (1 cluster each). Finally, subpopulations of B cells, neutrophils, basophils and cycling cells were also identified (1 cluster each).

We used for the first time in dogs the scRNA‐seq to investigate cellular subpopulations of the BALF. 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.